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  • Viskari, Ansa (2012)
    The purpose of this study was to investigate how the mixing time of the magnesium stearate affects on the compressibility of partially pregelatinized maize starch. Pregelatinized maize starch is used in pharmaceuticals as a filler, binder and as disintegrant. Because pregelatinized maize starch has lubricant characteristics itself, it is known to be sensitive for the amount and the mixing time of magnesium stearate. The aim is that magnesium stearate is not totally homogenously mixed on the powder surfaces so that even, clean powder surfaces exist. Homogeneous mixing means that particles are coated with magnesium stearate, which as a hydrophobic ingredient prevents bond formation between plastically and elastically behaving particles. Too much magnesium stearate and/or too long mixing time may cause weakening of tablet tensile strength, laminating and capping. The weakening of the tensile strength of the tablet increases friability, which causes problems during packaging process and the transportation. Too much magnesium stearate may also lengthen the disintegration time and slow down the dissolution. The aim of this study was to compare four different brands of pregelatinized maize starch. The purpose was to find differences affecting the compressibility behavior. Also the effect of the mixing time of magnesium stearate for compression behavior of masses were studied. The brands investigated were C*PharmGel DC 93000, Lycatab® C, Starch 1500® and SuperStarch 200®. First mentioned was a reference product which is not manufactured any more. There was only one batch of the reference product but three batches from other products to be able to investigate also batch to batch variation. The characteristics studied from pregelatinized starch samples were bulk density, apparent density and true density, flowability, moisture sorption, moisture content, pH value, swelling volume and particle size. Also NIR, FTIR and Raman spectroscopy and X-ray powder diffraction method were used. Weight, tensile strength, dimensions, friability, disintegration time and moisture sorption were studied for tablets. The compressibility of the mass and elastic behavior of tablets was studied. Pictures of the tablets were also taken by scanning electron microscope. When the mixing time of magnesium stearate was increased from 2 minutes to 5 minutes, the compression pressure needed for pressing tablets for 80 N strength increased 200-700 N depending on the brand of pregelatinized maize starch. Based on the results the best alternative to replace C*PharmGel DC 93000 was chosen to be SuperStarch 200®. Scanning electron microscope pictures showed that C*PharmGel DC 93000 deviates from other qualities studied by being roundish and regular in shape. SuperStarch 200® and Starch 1500® reminded remarkably each other. Lycatab® C was the biggest in particle size and very irregular in shape. The differences found in tabletting followed the expectations based on the SEM-pictures. SuperStarch 200® showed to best compressibility in lowest strain strength and after C*PharmGel DC 93000 it was least sensitive for mixing time of the magnesium stearate. It also has least elastic recovery. The differences between SuperStarch 200® and Starch 1500® in compression properties were moderate but clear. Lycatab® C had clearly the weakest compression properties.
  • Tervo, Annukka (2011)
    Alphaviruses are positive-stranded RNA-viruses and they belong to the family of Togaviridae. Alphaviruses are spread by mosquitoes of family Aedes. Alphaviruses have spread on all continents except Antarctica. So far 29 alphaviral species have been identified and they can be divided in two groups, Old and New world viruses, by their geographical distribution and by diseases they cause. Chikungunya virus (CHIKV) is one of the Old World alphaviruses and it has been found in Africa and Asia. However, due to the global warming, Chikungunya is also spreading to southern Europe. In humans, it causes fever, headache, rash and joint pain, which can last for several years and be very painful. In small children, Chikungunya can cause neurological symptoms such as encephalitis. Genome of alphaviruses encodes for four structural proteins and four non-structural proteins (nsP), of which nsP3 contains a macro domain. Macro domains are conserved in most kingdoms of life but their function has not been elucidated. It has been shown that macro domains bind ADP-ribose and its derivatives and it has been shown that nsP3-protein has an important role in alphavirus replication. The aim of the study was to study the use of compounds which bind to macro domain protein as antiviral agents. 45 compounds were chosen for antiviral studies by molecular modeling. These compounds were expected to bind to macro domain proteins. In a competitive binding assay five compounds inhibited more than 50 % poly-ADP-ribose (PAR) binding to MDO1-macro domain protein, which was the protein on which the molecular modeling was performed. When the competitive binding assay was performed with SFV macro domain (nsP3), only one compound inhibited poly-ADP-ribose binding more than 50 %. In SFV-antiviral assay seven compounds had inhibition percentage higher than 50 %. In a CHIKV replicon assay five compounds had more than 50 % inhibition on replicon expression. We also studied possible inhibition mechanism by studying whether the compounds inhibit the virus to enter the cell. Almost all compounds included in this assay inhibited the virus entry to some extent. In general, the inhibition of PAR binding and antiviral activity did not correlate among the studied compounds. Even though compounds which had antiviral potency did not inhibit PAR binding to macro domains, potential antiviral agents were found which deserve further investigation as virus entry inhibitors.
  • Anttila, Jenni (2013)
    Brain-derived neurotrophic factor (BDNF) and the receptor mediating its effects, neurotrophin receptor TrkB, seem to have a role in the pathophysiology and treatment of mood disorders such as depression and mania. BDNF is a neurotrophin that regulates the differentiation and survival of neurons and mediates neuronal plasticity. Lithium and valproate are mood stabilizing agents that are commonly used to treat mania but their mechanism of action is still unclear. However, both acute and chronic lithium treatment have been shown to activate TrkB receptor in the rodent anterior cingulate cortex. It has also been shown that chronic lithium and valproate treatment increase the amount of BDNF in the rodent brain. The aim of the experimental part of this master's thesis was to find out what are the effects of lithium and valproate on TrkB receptor activation and on the amount of intracellular BDNF protein levels in vitro on embryonic day 18 (E18) rat primary cortical neurons. In addition, the possible role of neuronal maturation was investigated by conducting the experiments with neuronal cultures aged 7 and 21 days in vitro. The research methods included two different types of enzyme linked immunosorbent assays (ELISA), phospho-Trk ELISA and BDNF ELISA. Western blot was used to confirm the results. Therapeutically relevant concentration of lithiumchloride and valproate blocked BDNFinduced TrkB receptor phosphorylation in immature neurons aged 7 days in vitro. The effect of valproate was detected only with ELISA. In contrast, therapeutically relevant concentration of valproate increased TrkB receptor phosphorylation in immature neurons after one hour treatment. Lithium and valproate did not regulate TrkB receptor phosphorylation in mature neurons aged 21 days in vitro. However, therapeutically relevant concentration of lithium increased BDNF protein content in mature neurons after 24 hours treatment. Therapeutically relevant concentration of valproate did not alter BDNF protein levels. In conclusion, neuronal maturation does have a role on the effects of lithium and valproate on TrkB receptor activation and regulation of BDNF protein levels. It is possible that lithium and valproate are harmful to immature neurons through blocking BDNF-induced TrkB receptor phosphorylation. Since therapeutically relevant concentration of lithium did not activate TrkB receptor as has been shown previously in vivo it seems that certain developmental processes are essential for lithium-induced TrkB receptor activation.
  • Qvist, Teo (2022)
    Ahmintahäiriö on epätyypillinen syömishäiriö, johon liittyy toistuvia ahmintakohtauksia, joiden aikana syödään hallitsemattomasti suuria määriä ruokaa, vaikka olo olisi jo epämiellyttävän täysi. Mantelitumake on ohimolohkon pohjukassa sijaitseva pelon ja tunteiden, mutta myös ruokahalun ei-homeostaattisen säätelyn, kannalta tärkeä rakenne. Mantelitumakkeen sentraalisen tumakkeen lateraalisen osan (CeL) solut säätelevät muun muassa ruoan palkitsevuutta ja tyydyttyneisyyden tunteen muodostumista ruokailun aikana. Suurin osa CeL:n kolinergisista hermoyhteyksistä on aivorungon pedunculopontisesta tegmentaalisesta tumakkeesta (PPT) projisoituvia hermoratoja. PPT:een kolinergisten hermopäätteiden in vivo optisen aktivaation on aikaisemmissa tutkimuksissa havaittu säätelevän opittua välttämiskäyttäytymistä, mutta optisen aktivaation vaikutusta ruoankulutukseen koe-eläimillä ei ole vielä tutkittu. Tämän pro gradu -tutkielman kokeellisen osan tavoite oli tutkia PPT:sta CeL:aan projisoituvan kolinergisen hermoradan in vivo optisen aktivaation vaikutusta ruoankulutukseen C57BL/6N -hiirillä. Optisen aktivaation vaikutusta tutkittiin sekä homeostaattiseen että ei-homeostaattiseen ruoankulutukseen. Ei-homeostaattisen ruoankulutuksen tutkimiseksi hiirille indusoitiin ahminnan kaltaista syömiskäyttäytymistä tarjoamalla hiirille rasvapitoista ruokaa viikoittain 24 tunnin jaksoissa. Tämän lisäksi tutkittiin ovatko optisen aktivaation vaikutukset kumottavissa asetyylikoliinireseptorien antagonistien mekamyyliamiinin tai skopolamiinin intraperitoneaalisella annostelulla. In vivo optisella aktivaatiolla ei havaittu olevan tilastollisesti merkitsevää vaikutusta ruoankulutukseen C57BL/6N -hiirillä. Tästä johtuen myöskään asetyylikoliinin antagonistien annostelun vaikutusta optisen aktivaation vaikutuksiin ei voitu arvioida. Tulokset viittaavat siihen, että PPT:een ja CeL:n välinen kolinerginen hermorata säätelee koe-eläimillä opittua välttämiskäyttäytymistä, mutta ei ruokahalua. Näin ollen on myös epätodennäköistä, että optisen aktivaation vaikutukset edellyttävät CeL:n proteiinikinaasi C deltaa ilmentäviä soluja. PPT:een kolinergisten hermosolujen vaikutus CeL:n toimintaa hermosolutasolla tulee kuitenkin varmistaa tarkemmissa jatkotutkimuksissa. Vaikka tutkimuksessa ei havaittu tilastollisesti merkitsevää vaikutusta, havaintoihin on kuitenkin suhtauduttava varauksella, sillä tutkimuksen toteuttamiseen liittyneet haasteet voivat rajoittaa havaittujen tuloksien luotettavuutta.
  • Engström, Isanora (2023)
    The long-term use of antidepressants has increased significantly worldwide in recent decades. Deprescribing and the expertise related to it is an important part of the individual drug treatment optimization, the management of long-term diseases, the avoidance of adverse drug effects and the improvement of treatment outcomes. The aim of this thesis was to examine the information found in the statutory Summary of Product Characteristics (SmPC) and other key information sources for healthcare professionals about antidepressant deprescribing. A qualitative content analysis was conducted on SmPC (n=15) of the antidepressants (escitalopram, mirtazapine, sertraline, citalopram, venlafaxine) selected for the study, three national depression treatment guidelines (Suomalainen Lääkäriseura Duodecim: Depressio Käypä hoito -suositus, American Psychological Association APA: Practice Guideline for the Treatment of Patients with Major Depressive Disorder, United States and National Institute for Health and Care Excellence NICE: Depression in Adults: Treatment and Management, United Kingdom) and one decision supporting deprescribing tool (MedStopper). The content, quantity, and quality of information about antidepressant deprescribing varied between the information sources included in the study. However, the information found in the SmPC and the MedStopper -tool was mostly in line with the information found in the clinical practice guidelines included in the study. Most general information about antidepressant deprescribing or measures that can be used to guide deprescribing was found in the clinical practice guidelines. In all examined sources, antidepressants were recommended to be discontinued in a controlled manner by gradually reducing the dose. However, the recommended duration of the dose reduction varied in different information sources. A detailed dose reduction program was not found in most of the information sources. A detailed dose reduction program was found in only one clinical practice guideline (NICE) and the MedStopper -tool. The continuation of antidepressant treatment after remission and the timing of stopping the medication was discussed in only two clinical practice guidelines (APA and Käypä hoito). However, instructions for action if severe or intolerable discontinuation symptoms appears were found in almost all information sources. Only the clinical practice guidelines mentioned the recurrence of depression as a possible harm when stopping the medication and instructed how to act in the event of a possible relapse. Benefits related to antidepressant discontinuation was not mentioned in any of the examined information sources and only one clinical practice guideline (NICE) discussed barriers related to stopping antidepressants. The information found in individual information sources was insufficient and provided little support for healthcare professionals to guide deprescribing. Current key sources of information for healthcare professionals provide limited information and relatively imprecise guidance on antidepressant deprescribing and how to support the antidepressant discontinuation process. Better randomized clinical trials are needed to develop clearer and more extensive evidence-based guidelines for healthcare professionals on antidepressant deprescribing and to prevent unnecessary long-term antidepressant treatment and patient exposure to possible adverse drug effects.
  • Kamppari, Emilia (2015)
    Both primary pharmacokinetic (PK) parameters, volume of distribution (Vd) and clearance (CL), undergo considerable developmental changes in infancy and childhood, necessitating compensatory changes in dosing regimens if therapeutic effect without toxicity is to be reached and maintained. Neonates exhibit higher body water content and decreased plasma binding capacity compared to adults, producing increased Vd values for many drugs. Due to immaturity of glomerular function and low metabolic enzyme expression, CL tends to be significantly decreased in neonates. Both Vd and CL undergo simultaneous but independent maturation during development. Performing pediatric clinical trials is challenging due to ethical and practical constraints. Modeling and simulation approaches, such as population pharmacokinetic (POP-PK) and physiologically based pharmacokinetic (PBPK) modeling, are beneficial aids in planning and performing clinical studies in children. The aim of the literary review is to assess the developmental phenomena that cause pediatric pharmacokinetics to differ from adults, the clinical consequences arising from these differences, and present ways to apply POP-PK and PBPK models in pediatric drug research. In the experimental work, two modeling approaches for the prediction of pediatric pharmacokinetics are explored. First, the performance of the commercial PBPK software Simcyp in simulating a drug-drug interaction between cyclosporine A (CsA) and ketoconazole (KTZ) is assessed. Second, a method for in vitro-in vivo extrapolation (IVIVE) of CL in children is developed and evaluated. The aim is to assess the suitability of both modeling methods in pediatric drug research. Simcyp predicted the general age-related trends in the CsA CL and CsA-KTZ interaction well for the most part. However, the values of the simulated CL terms and magnitude of the simulated interaction were significantly under- and overpredicted, respectively. Due to limited clinical data, though, Simcyp performance could not be fully validated. The method developed here for IVIVE of pediatric CL values yielded successful predictions in most cases, with in vitro data from hepatocytes performing slightly better when compared to microsomal data. Success in extrapolations performed for adults correlated well with success in the pediatric extrapolations. Therefore, in drug development, the method developed in this work would be most useful after adult PK data is available, before the first pediatric clinical studies.
  • Niklander, Johanna (2018)
    Tämä tutkimus esittelee kasviperäisen nanokuituselluloosageelin (NFC; GrowDex®) arvioinnin kolmiulotteisena (3U) kasvualustana rintarauhasen organogeneesin mallinnuksessa. Tutkimuksen tavoitteena oli tarkastella kasviperäisen in vitro -kasvualustan aiheuttamaa solusäätelyä normaalissa rinnan epiteelisessä solulinjassa, sekä selvittää rintakudoksen rauhasrakenteiden muodostumisessa keskeisen laminiini 111:n (LAM-111) alustaan lisäyksen mahdollisia hyötyjä viljelmille. Tutkimuksen koeasetelmassa NFC:n edustamaa kasvunicheä arvioitiin ihmisen rintaepiteelistä eristetyllä -ja tyvikalvon proteiinikontaktien säätelystä riippuvaisella MCF 10A -solulinjalla. Solujen in vitro -nicheympäristön verrokkimallinnuksessa hyödynnettiin epiteelisen tyvikalvon proteiiniympäristöä edustavaa proteiinirikasta Matrigel™-2,5U -kasvualustaa. Viljelynäytteistä tehtiin aikapisteittäin valomikroskooppiset -sekä histologiset hematoksyliini – eosiini (HE) morfologian arvioinnit, e-kadheriinin, vimentiinin ja β4-integriinin ilmentymisten vasta-aine-analyysit, sekä β1-integriinin, Bim:in ja c-FLIP-L:n lähetti-RNA:n reaaliaikaiset PCR-analyysit. Analyyseissä keskityttiin tarkastelemaan rintarauhasen epiteelin polarisoitumistapahtumassa havaittavaa solusäätelyä ja proteiinien eritystä. LAM-111 -lisän havaittiin edistävän jossain määrin NFC:ssä viljeltyjen sferoidien sisämorfologian kavitaatiota sekä eritettyjen proteiinien sijoittumista sferoidien pintarakenteisiin Matrigel™ -kontrollinäytteiden kaltaisesti, muttei yksinään riittänyt tuottamaan Matrigel™ :ssä havaittua viljelmien homogeenisyyttä. Kokeen natiivi-NFC:ssä sekä NFC-LAM-111:ssä kasvaneiden sferoidien PCR-analyyseissä havaittiin polarisaatiotapahtumaan liittyvää solusäätelyä viljelmien loppuvaiheessa päivänä 28, poiketen vastaavan PCR profiilin ilmentymisestä Matrigel™ -viljelmissä jo päivänä kolme. NFC -olosuhteissa havaittiin myös Matrigel™ -viljelmistä puuttuvia ylimääräisiä, epiteelisiltä vaikuttavia rakenteita, joiden määritteleminen vaatii lisätutkimuksia. NFC todettiin jäykkyyden suhteen helposti muokattavaksi sekä mahdollisesti kudoksen mekaanisia ominaisuuksia jäljitteleväksi 3U -kasvualustaksi. Tämän kokeen tuloksien perusteella muokkaamatonta NFC:tä voidaan ehdottaa soveltuvaksi kasvualustaksi tyvikalvoproteiinien säätelystä riippumattomille solutyypeille, sekä solutyypeille, jotka kykenevät tuottamaan ympärilleen oman kudostyypillisen proteiiniympäristönsä. Kliiniseen käyttöön kelpuuttavat standardivaatimukset täyttävä NFC vaikuttaa lupaavalta materiaalilta räätälöitävien in vitro -kasvualustojen suunnitteluun, ja mahdollisesti tarjoaa rakenneosiltaan tarkasti määritellyn, xenovapaan, ja proteiinilisillä eri solutyypeille säädettävän in vitro -kasvunichen tulevaisuuden jatkotutkimuksiin.
  • Mandelin, Ronja (2024)
    MDMA is an illegal stimulant known for its empathy-enhancing effects. Its positive effects are mainly based on increasing the concentrations of monoamines such as serotonin (5-HT), dopamine (DA) and norepinephrine (NE). In addition to its positive effects, MDMA can cause adverse effects such as hyperthermia and neurotoxicity. Especially with long-term use, MDMA can cause serotonergic and dopaminergic neurotoxicity. In addition, there are also indications of MDMA-induced neurotoxicity in systems where gamma-aminobutyric acid (GABA) functions as the main neurotransmitter. Glutamate decarboxylase (GAD) 67 is an enzyme that synthesizes GABA from glutamate and is a specific marker for GABAergic cells. The amygdala is a nucleus in the brain that regulates anxiety and fear response. In addition to GABAergic interneurons, there are also glutamatergic cells in the basolateral nucleus (BLA) of the amygdala, however in the central nucleus (CeA) there are only GABAergic cells. Disturbances in the GABAergic system can predispose to psychiatric diseases such as anxiety. The aim of thisstudy was to investigate the effects of MDMA (20 mg/kg) on the number of GAD67-positive cells in two nuclei of the mouse amygdala, BLA and CeA. In addition, this study aimed to examine the importance of the dose (4 or 16 injections) for neurotoxicity and the duration of the effects (2, 7 or 30 days). Adolescent wild type mice were divided into 12 groups according to the treatment (MDMA or saline), dose and timepoint. After euthanasia, the brain sections at the level of the amygdala were collected and stained with an immunohistochemical method and imaged using a confocal microscope. This study showed that MDMA reduced the number of GAD67-positive cells in the BLA when mice were given a total of 4 injections. This effect lasted up to 30 days. In contrast, MDMA did not reduce the number of GAD67-positive cells in the BLA in mice that were given 16 injections. Also, MDMA did not decrease the number of GAD67-positive cells in the CeA, regardless of dose. Statistical significance could have been improved, for example, by using more mice or analysing more sections from each individual animal. It is important to continue studying the effects of MDMA to better treat and prevent its adverse effects. In addition, increased understanding would urge users to exercise caution when using MDMA.
  • Koskenkorva, Tiina (2012)
    Elucidation of transporter- and/or metabolic enzyme-mediated drug interactions is important part of early drug development. However the knowledge about clinical consequences of transporter-mediated drug-drug interactions is still limited and more investigation is needed to improve our understanding. MDR1 transporter, widely distributed on the pharmacokinetic barriers in the body (e.g. intestine) and has been shown no limit the bioavailability of drugs. Substrates of MDR1 are exposed to limited intestinal drug absorption and intestinal drug-drug interactions due to inhibition of the transporter. In predicting the clinical significance of an interaction, the principal obstacle has been the limited ability to appropriately scale the preclinical data into in vivo situation. In vitro-in vivo correlations on the extent of MDR1's influence on absorption and standardized predicting methods for drug-drug interactions using the inhibitory constants (IC50 and Ki) would greatly increase the value of in vitro studies. Current in vitro and in silico methods for prediction of the influence of MDR1 on intestinal absorption and related drug-drug interactions are discussed in the literature review. In addition, the latest regulatory draft guidances (FDA, EMA) are reviewed. Aliskiren has been shown to be a sensitive MDR1 substrate in vivo and high affinity substrate for the transporter in vitro. The objective of the experimental work was to study the MDR1-mediated transport of aliskiren and the related drug-drug interactions in vitro and in silico. Vesicular transport assay was used to obtain kinetic parameters for aliskiren (Km and Vmax) and inhibitor potencies (IC50) for ketoconazole, verapamil, itraconazole and its metabolite hydroxyitraconazole. Ki was further calculated for itraconazole and hydroxyitraconazole. Aliskiren showed high affinity to MDR1 transporter with a Km value 5 µM, consistent to what was reported previously in different assay systems. The interactions between aliskiren and the inhibitors in vitro correlated to the observed interactions in vivo in humans. In addition, hydroxyitraconazole was shown to be a potent inhibitor of MDR1-mediated transport of aliskiren in vitro. This suggests that hydroxyitraconazole may contribute to the pronounced interaction observed between aliskiren and itraconazole in a clinical interaction study. A compartmental absorption and transit (CAT) model with added enterocyte compartments and MDR1 efflux was used to describe the influence of MDR1 on intestinal absorption of aliskiren in humans. The integration of kinetic parameters (Km) from in vitro studies requires further optimization on how to describe the intracellular drug concentrations in the model. Aliskiren is however suitable MDR1 probe substrate to be used in in vitro and in vivo trials in humans and therefore gives a good basis for developing vitro-in vivo predictive models.
  • Tepponen, Tuomas (2017)
    Multidrug resistance protein 1 (MDR1, p-glycoprotein) belongs to the ATP-binding cassette transporter family and it's encoded by ABCB1/MDR1 gene. It is a protein which transports many different kinds of compounds out of cells, for example from endocytes to the lumen with the use of energy from ATP. MDR1 is there for a restrictive factor for several orally administered drugs. It`s important to have knowledge about MDR1-inhibitors, in order to avoid harmful drug-drug and food-drug interactions that might affect medical treatment. The purpose of this master's thesis was to optimize an in vitro MDR1-vesicle uptake method and use it to screen inhibitors from compound libraries. To optimize the method, the effect of cholesterol loading on ATP-dependent transport of test substrate N-methylquinidine (NMQ) was evaluated, transport kinetics of the vesicles and kinetics of known inhibitors were also tested. With the optimized method, screening was done with a library of 25 food additives and a library of 42 synthetic compounds. The chemical structures of the synthetic compounds were analyzed manually in order to find factors that could explain their ability to inhibit MDR1. Only one inhibitor was found among food additives: curcumin. Other additives didn't increase or decrease the ATP-dependent transport of NMQ. Several inhibitors were found from the library of synthetic compounds, also a couple of compounds were found to increase the active transport of NMQ. Results indicate, that the additives used in this study have low risk to cause MDR1 mediated interactions, if curcumin is excluded. The inhibitory effect of curcumin should be investigated in in vivo-situation, because vesicle-based in vitro-results have tendency to overestimate results. Screening results of the synthetic compounds gives more confirmation to the usefulness of the screening method. The MDR1-inhibition screening method described in this Master`s thesis is valid, and it can be used to screen different compound libraries for MDR1-inhibitors. In the future it could be used to screen different kinds of compounds, which might end up inside humans and cause interactions with drugs.
  • Puustinen, Sanna (2011)
    Drug-drug interactions occur when a drug or a drug metabolite modifies the activity of a drug metabolizing enzyme. As a result the concentration of active drug can be too low to be effective or too high and possibly toxic. This is an increasing problem in drug therapy where polypharmacy is rather common today. Therefore, in drug discovery and development significant efforts have been made in order to predict such interactions in advance and avoid them, or at least minimize them. This study is focused on medetomidine, a drug metabolized by UDP-glucuronosyltransferases (UGT). The aim of the study was to find inhibitors for medetomidine glucuronidation. Also the mechanism of possible inhibition was of interest. It is already common to test interactions of a given enzyme substrate with other enzymes of the same family either in phase I or phase II of drug metabolism in humans. It is less common, however, to examine such interactions between enzymes of two different families. In the present study it is tested if the compounds which are possible inhibitors of cytochrome P450 monooxygenase (CYP) also inhibit UGTs. Inhibition of glucuronidation was studied with HPLC method previously developed for medetomidine glucuronidation. First glucuronidation of medetomidine was studied without inhibitor compounds. After that the impact of three possible inhibitors on medetomidine glucuronidation was studied and results were compared with the initial results. Three compounds were found to inhibit glucuronidation of medetomidine. Also an interesting change in UGT's enzyme kinetics after the binding of inhibitor was discovered. It is interesting that same compounds could inhibit both CYPs and UGTs. The results revealed that if a CYP and a UGT could bind for the same compound, it is also likely that structural analogues of that compound will interact with both enzymes. In drug discovery and development it is important to take into account both CYP-enzymes and less studied UGTs, and their possible interactions.
  • Jansson, Teresa (2019)
    Conditional reimbursement was introduced in Finland in January 2017 as a temporary addition in the Finnish Health Insurance Act. An agreement can be made between a marketing authorisation holder (MAH) and the Pharmaceuticals Pricing Board. Conditional reimbursement status can be allowed for a medicinal product if the drug is addressing unmet medical need and there are uncertainties associated to the medicinal product considering i.e. therapeutic value or cost-effectiveness, when traditional reimbursement procedures are not suitable. Risk-sharing is an essential part of the agreements and the results are monitored. Types of agreements are divided into financial- and performance-based agreements. Conditional reimbursement in Finland has not yet been studied in a large extent since its introduction. The aim of this study was to create an overview of the medicinal products with conditional reimbursement in Finland, how the unmet medical need is addressed, and which treatment options are available. Also, benefits and risks of the different stakeholders of risk-sharing agreements (RSA), why these agreements are worth to implement, earlier experiences from the European Economic Area (EEA) countries and what pharmaceutical companies should consider prior to negotiations were investigated. A document analysis was performed for investigating the medicinal products with conditional reimbursement status in Finland. A systematic literature review was conducted for collecting information and earlier experiences of RSAs and managed entry agreements (MEA) in the EEA-countries. On February 1st, 2019 there was 19 medicinal products with conditional reimbursement in Finland. These drugs are successfully addressing unmet medical need. All stakeholders of RSAs encounter benefits and risks of these agreements but the MAH is the one carrying the largest responsibilities and risks. Risk-sharing agreements gained in popularity since the early 2000s in the EEA-countries. There is no golden standard for types of agreements made but MEAs are enshrined in legislation in most countries. The pharmaceutical company should as early as possible start shaping details and collect information of the product for which conditional reimbursement will be proposed to. Negotiations might be challenging, but the aim is an agreement in which both the MAH and the payer are content with. Finland is following a similar trend as other EEA-countries, since most of the medicinal products with conditional reimbursement are oncology medicines. The use of the drugs has been limited through reimbursement number codes for certain patients who are most likely to benefit from the treatment. Rationales for introducing RSAs in EEA-countries were similar, e.g. working with finite resources, improving access, reducing uncertainty and prices, managing budget impact and improving cost-effectiveness. It seems like Finland is unique by the temporary introduction of conditional reimbursement in legislation and in other countries it has been introduced as permanent. Starting the preparations early for negotiations could save time and resources. When a RSA is made and the medicinal product shows the benefits expected, this is the ideal situation where all stakeholders benefit.
  • Ukkonen, Anni (2020)
    The package leaflet (“leaflet”) is a technical document included in medicine packages to provide information about the medicinal product to the user. With the EU now encouraging the adoption of eHealth, it can be assumed that written medicine information would be included in the digitalisation process. Medicine users’ views on electronic forms of medicine information should be assessed before any changes can be made, but so far there is very little data on this. The aim of this study was to find out what kind of leaflet medicine users would prefer and how they would feel about an electronic leaflet. The main aim was to find out if there is a difference in preferences between different types of medicine users and between medicine users of different ages in the provision of a package leaflet. The study also sought to find out if the current leaflet is being read by medicine users. This study was conducted by carrying out a survey to pharmacy customers over the age of 16 collecting prescription medication(s) for themselves (n = 110). The data was collected at one retail pharmacy in Helsinki, Finland during July 2020. The data was analysed quantitatively. This study found that medicine users generally feel positively about an electronic leaflet (liked by 63%) and many are open to idea of an electronic leaflet (75%). The majority (88%) could see positives in using an electronic leaflet, regardless of leaflet preferences. The study did not find a difference between new and repeat medicine users in the preference for a particular leaflet format, but age is correlated with the preference for a particular leaflet type, with younger medicine users wanting an electronic leaflet as often as older medicine users want a printed leaflet. Having the leaflet appear in My Kanta pages after the medication has been dispensed was found to be the most popular way to receive an electronic leaflet. This study also found that there is a difference between new and repeat medicine users when it comes to reading the leaflet after a medication has been dispensed. With the current printed leaflet 81% of repeat medicine users and 38% of new medicine users do not read it. The most common reason given for not reading the leaflet was that the participant had read it before and did not feel the need to read it again. According to this study, medicine users, especially younger medicine users, feel positively about the idea of an electronic leaflet, which is encouraging for the future of an electronic leaflet. The results are in line with prior research, but also suggest that more medicine users feel positively about the idea of an electronic leaflet than before. The leaflet reading behaviours of medicine users also highlight the need for a system, where a medicine user can be alerted to any changes in the leaflet, which is something only an electronic system could do.
  • Kainulainen, Tuija (2015)
    The significance of OTC product sales has risen in pharmacies because of lower margins obtained from medicines and thus a fall in the revenue. Manufacturing enterprises must pay particular attention to the success of product launches to ensure that their products end up on pharmacy shelves instead of competitors. The study intended to determine if the known key factors of successful launch also apply when launching a product to pharmacy market and if any of these factors was thought to be the most important one from pharmacists' perspective. In addition it was researched if there would be some important factors to be considered exclusively in product launches to pharmacy market and which factors have the greatest impact on pharmacies decision making about the product selection. The study was conducted as a survey directed to pharmacists, in which just launched D-vitamin product Elivo Vahva+ D50 was used as an example product. As a second part of the study few participants were interviewed by e-mail. According to the study pharmacies are interested in products that fit their selection, in other words, they are proven to be effective and useful for customers. They should bring some added value to the existing selection, to be visually attractive, price-reasonable and with a large enough target group. Representative visits, product visibility in the media, as well as the customers demand have the greatest impact on pharmacies decision making about which products to include to the selection. In addition, belonging to a pharmacy chain often brings with it the obligation to keep certain products in the shelves. Least impact on the decision making was with the electronic newsletter and pharmacy events. Pharmacies profit margin, as well as the possible purchase discounts and OTC products compensation practices are also taken into account in selection decisions. It is important that the company invests in their representatives education and offer reliable product knowledge and sales arguments to pharmacies for example with personnel training or at least in the form of brochures. When deciding the timing of the launch, seasonal variations in sales as well as competitors market entries needs to be taken into account. If it's not possible to be the first in the market, the product needs to have a real added value compared to others.
  • Piipponen, Anu (2016)
    Pharmaceutical nanocrystals are under one micrometer sized crystals composed of pure active pharmaceutical ingredient (API) and stabilizer. Their apparent dissolution rate is improved compared to conventionally sized crystals. Rapid dissolution is mainly due to increased intrinsic surface area of API powder. Solubility increase is significant only with very small, under 100 nm crystals. Nanocrystal formulations with improved dissolution rates can be utilized to increase bioavailability of fairly insoluble BCS class II APIs. Few nanocrystal based products are already on market. Common methods for dissolution study of nanocrystals arecompendial dissolution apparatus 1 or 2, which usually rely on sampling and separation of undissolved fraction. The reliability of these methods is dependent of the separation efficiency. Unfortunately separation becomes more tedious with diminishing crystal size. Thus it would be desirable to replace the methods that require sampling and separation with methods that do not require separation of undissolved fraction (in situ methods), preferably with continuous detection. With the dialysis method the separation is easily achieved. However, the rate limiting step is not dissolution but diffusion through the dialysis membrane. Electrochemical in situ detection methods can only be applied to electroactive APIs. Utilization of in situ UV probes for monitoring nanocrystal dissolution is limited by the UV absorbance of the nanocrystals themselves. To date, light scattering methods have mainly been applied to solubility studies, with few attempts on dissolution studies. In this study the light scattering, dialysis and compendial paddle methods were compared for their ability to monitor the dissolution of indometacin nanosuspensions (NS). Light scattering experiments were performed with Zetasizer equipment. Three poloxamer 188 stabilized NSs, with average diameters (Dz) of 300 nm, 600 nm, and 900 nm, were evaluated. Dissolution studies were executed in sink conditions (under 30% of saturated concentration) and in slightly higher concentration (intermediate conc., 30-50% of saturated concentration) at pH 5.5. The compendial paddle method was performed on the same suspensions with the same medium at intermediate concentration. In the dialysis method the studied NS had a Dz value of 350 nm. The pH of the dissolution medium was 7.4, and the membrane was made of regenerated cellulose. Experimental results were fitted to exponential equation and the dissolution time DT, i.e. time to reach 99% dissolution, was determined based on the equation. In sink conditions the dissolution of all of the NSs was so rapid that reliable estimations of dissolution times could not be made with the light scattering method. In intermediate concentration the dissolution time (51±12 s) of the 300 nm NS was significantly lower than those of 600 nm (340±80 s) and 900 nm (230±50 s) NSs with a confidence level of 5%. The slowest dissolution of the 600 nm NS could be attributed to its broad crystal size distribution. With the compendial paddle method no significant differences in dissolution times could be detected. Compendial dissolution times, about 600-700 s, were markedly longer than those from light scattering experiments. The dialysis method was unable to discriminate between 350 nm NS and indometacin solution, which can be explained by rapid dissolution of the nanocrystals, followed by slow diffusion across the dialysis membrane. Of the studied methods, light scattering was the only one to discriminate between dissolution times of various NSs. It was most applicable to narrow crystal size distributions. It is a fairly small scale method requiring only 1 mL of dissolution medium and about 10 µg of nanocrystals. The method was not dependent on chemical analysis. Theost important limitation was the fact that due to the operational method of the Zetasizer, the first data point was not acquired until about 20 s after the measurement started.
  • Bruun, Tanja (2018)
    Marine organisms can be regarded as a diverse source of bioactive compounds with the possibility to discover novel drug lead molecules. Sea sponges produce bromine containing alkaloids, bromotyrosines, from which several are active against cancer. Some bromotyrosines have spirocyclic structure and the innate three-dimensionality and structural novelty of spirocycles make them an interesting option in drug design. Clavatadine C, extracted from sponge Suberea clavata, is a bromine containing spirocyclohexa-dienylisoxazoline alkaloid. It’s symmetric spirocyclic core can be viewed as a restricted derivative of open chain oximes, such as purpurealidin I, a bromotyrosine extracted from Pseudoceratina purpurea. Earlier work with purpurealidin I derivatives against melanoma cell line has had some promising results. Inspired by these earlier results, eight spirocyclic clavatadine C derivatives were synthesized according the published synthesis route. The activities of seven synthesized clavatadine C derivatives were tested on A375 melanoma cell line. All spiro derivatives were active with CC50 values ranging between 1.0 μM and 3.4 μM. Also, the activities of 10 earlier synthesized bromotyrosine derivatives were tested, from which four open chain oximes had CC50 values between 13.5 μM and 27.8 μM. Interestingly, the most active compounds were chlorinated and unhalogenated spirocyclic derivatives. In general, the spirocyclic compounds were 2- to 8-fold more active than the corresponding open chain oximes. The selectivity of active compounds was determined as cytotoxicity against Hs27 fibroblasts and by comparing the CC50 values of these two cell lines. The most selective compound was brominated derivative which had three times better selectivity against melanoma cells. The weak selectivity was consistent with the trend with open chain oxime analogs. Despite the selectivity issue, the improved activity of spirocyclic derivatives are promising and support for further investigation of marine-based spirocyclic bromotyrosine derivatives against melanoma.
  • Mankila, Anja; Mankila, Anja (2022)
    Cardiovascular diseases are the most common causes of mortality worldwide. More adequate human-based models would be needed for the purposes of disease modeling and drug development. One of the most promising fields of in vitro modeling is the use of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). A central problem of hPSC-CMs is their immature or fetal-like phenotype compared to adult human cardiomyocytes regarding many structural, functional, and metabolic properties. The development of metabolic properties is considered to be a central driver of cardiomyocyte maturation. One practicable way to promote the metabolic maturation of hPSC-CMs in vitro is the use of various biochemical cues in the cell culturing media. The topic of this study was the metabolic maturation of hPSC-CMs. The research questions were: What biochemical cues have been suggested to be involved in the hPSC-CM maturation in vitro? What signaling pathways connected to the biochemical cues have been explored in the context of the maturation of hPSC-CM? What experimental results have been achieved on the effects of the biochemical cues and the involvement of the signaling pathways? The study was conducted as a systematic review with the database Scopus (Elsevier). The final set of materials consisted of 46 original research articles published in peer-reviewed journals in English in the years 2013–2022. Out of the materials, 11 articles (24%) were characteristically longitudinal studies. They indicated that the pathways leading to metabolic changes such as PPARs (peroxisome proliferator-activated receptors) and PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α) are activated already in early stages. In 12 articles (26%), pharmacological agents were used to target the metabolic pathways, and in 8 articles (17%) techniques affecting the gene expression were utilized. The most recent studies involved ever more frequently combinations of different techniques. Considering the use of biochemical cues, the trend has been to favor fatty acids, thyroid hormone and dexamethasone over glucose, insulin and insulin-like growth factor. Some cues such as retinoic acid and neuregulin 1 have been tested only in single experiments. In addition to the nuclear receptor mediated pathways, the energy sensors AMPK (AMP-activated protein kinase) and mTOR (mechanistic target of rapamycin), the oxygen sensor HIF-1α (hypoxia-inducible factor 1α), and the microRNAs turned out to be central.
  • Parviainen, Heli (2020)
    Statins are a commonly used group of drugs that reduce the cholesterol levels in blood and have been shown to reduce cardiovascular morbidity and mortality. However, a considerable percentage of patients experience adverse effects during statin treatment. Statin adverse effects have been associated with genetic polymorphisms and drug-drug interactions that affect the elimination and active transport of these drugs. A more comprehensive knowledge of statin metabolism may be a step towards better management of statin treatments. Statin metabolism both in vivo and in vitro has been subject of study for years. In vitro incubation conditions may considerably affect the observed clearance, and results obtained with different methods or in different laboratories may not be directly comparable to each other. No single in vitro study on a wide panel of statins has previously been conducted. Six statins and some of their metabolites, fourteen compounds in total, were included in the study. The intrinsic clearance (CLint) of these molecules was investigated in vitro on human liver microsomes (HLM) and a panel of eleven cytochrome P450 (CYP) enzymes recombinantly expressed in E. coli. Observed CLint values for each compound in HLM and for each compound-CYP pair with observed depletion were calculated. The percentual contributions of each CYP enzyme to the metabolism of the compounds was calculated. The results obtained with recombinant CYP enzymes (rcCYP) were complemented with studies on HLM with specific chemical inhibitors of CYP enzymes. In this study the metabolism of statin lactones seemed to be faster than the metabolism of the corresponding statin acids. Atorvastatin lactone, 2-hydroxy atorvastatin lactone, 4-hydroxy atorvastatin lactone and simvastatin were extensively metabolized. Atorvastatin, 2-hydroxy atorvastatin, 3R,5S-fluvastatin, 3S,5R-fluvastatin, pitavastatin lactone and simvastatin acid showed intermediate metabolism. 4-hydroxy atorvastatin, pitavastatin, pravastatin and rosuvastatin rates of metabolism were below quantification limit. CYP3A4 had a major role in the metabolism of atorvastatin and its metabolites, simvastatin and simvastatin acid. CYP3A4 also had activity towards pitavastatin lactone. CYP2C9 had a high activity towards both 3R,5S-fluvastatin and 3S,5R-fluvastatin. CYP2D6 may play a part in the metabolism of pitavastatin lactone. CYP2C8 may have some activity towards simvastatin and simvastatin acid. The data is mostly in agreement with previous in vitro and in vivo studies regarding both the metabolism rate of statins and the contributions by different CYP enzymes to the metabolism of statins. Due to the screening nature of the study and some methodological constraints, these data should be considered as preliminary and require confirmation in further studies.
  • Hyttinen, Nea (2023)
    Chronic wounds are a worldwide health problem that produce a lot of costs for society and can have a substantial impact on patients’ quality of life. Human adipose stem cells (hASCs) have been studied as a treatment option for chronic wounds as they can induce wound healing in many ways. Extracellular vesicles (EVs) produced by hASCs are a great solution to acquire the benefits of hASCs while avoiding their problems such as possible mutagenicity. HASC-EVs have been found to induce wound healing by for example enhancing angiogenesis and fibroblast proliferation. HASCs can be grown in 2D where the cells attach to the bottom of the cell culture vessel or in 3D where the cells attach to each other and create a spheroid. 2D cell culturing is easy and inexpensive but 3D cultured cells resemble in vivo –like conditions more. Because of these in vivo -like features, hASCs grown in 3D might produce EVs that resemble the properties of host cells in natural environment more than 2D. The aim of this thesis was to compare 2D culture, matrix-based nanofibrillar cellulose (NFC) hydrogel culture, and matrix-free suspension culture in ultra-low attachment (ULA) wells as growing platforms for hASCs and as continuous EV production methods. During culturing, the conditioned media was collected after which, the EVs were isolated, and the EV concentration and size range was measured with nanoparticle tracking analysis (NTA). After culturing, the metabolic activity of hASCs was measured and the cells were collected for immunocytochemistry (ICC) assay, western blot (WB) assay, and for quantitative PCR (qPCR) to examine the stemness and differentiation of hASCs grown in different cell cultures. The hypothesis of this thesis was that the NFC cell culture would produce the best EV yield and the best EVs for therapeutic use. Based on the acquired results, this hypothesis could not be supported. When visually inspecting the cells, all three cell cultures were viable but the metabolic activity of hASCs in NFC hydrogel was low compared to 2D and suspension cultures. Also, the EV, protein and RNA yield were lower in NFC. ICC, western blotting, and qPCR results were inadequate to make a straightforward implication of what cell culturing condition is the best for EV production and they would need repetition and optimization. Looking at the overall results, 2D cell culturing produced the best EV and RNA yield, had the highest metabolic activity and was least laborious cell culturing method which makes it a good option for continuous EV production. Suspension culture on the other hand resembles in vivo -like environment which could possibly produce better EVs for therapeutic use. The metabolomic assays on the EVs would be interesting to perform in the future to examine if the in vivo –like features affect the quality of EVs.
  • Räisänen, Titvi (2023)
    A clean area is an area isolated from its environment to prevent contamination of final product during aseptic processing. The clean area can be divided into four different grades from A to D, which all have different cleanliness standards. Grade A is the highest grade where preparing products that are not terminally sterilized must be performed. Airlocks are located between different grades to prevent free airflow, and enable necessary precautions, such as putting on protective garments and disinfecting material surface. These procedures reduce risk of contamination of the higher grade. The purpose of this study was to create a protocol to help evaluate material disinfection and transfer processes in the hospital pharmacy of Turku University Central Hospital and to determine surface bioburden of material stored in the grade C area. Surface samples of the examined material were taken in accordance with in-house guidelines by using contact plates and swabs depending on the surface of the material examined. After incubation, colony forming units were counted. Samples were taken from primary packages of ingredients and equipment stored in grade C area, as well as from material transfer boxes and cut flush plastic folders used in the clean area. Samples were taken both before and after routine disinfection of this material. 45 % of the samples taken before disinfection were contaminated. The lowest contamination rates were observed from items made from glass and those that were stored in their secondary package. In five plates grew more than 25 colonies, of which two had biofilm covering the whole surface of the plate. These samples were taken from larger plastic items, such as an infusion bag and a plastic folder. High bioburden is possible on the surface of material stored in grade C clean room, despite precautions. 25 % of the samples taken after routine disinfection were contaminated with a maximum of two colonies per plate. Despite disinfection, viable microbes may remain on the surface of material. Material with risk of high bioburden were selected for the protocol. Items were disinfected and transferred to grade B area as a simulation of normal processes. Different operators performed the protocol a total of eight times. 14 % of samples were contaminated with a maximum of two colonies per plate, except for one plate with 15 colonies. This repetition exceeded the limits set for the protocol. One repetition had zero contaminated samples. The bioburden of material surface after disinfection is affected by operators, cleanliness of the grade C area, and manipulation of the storage. A high bioburden increases risk of unsuccessful disinfection, and recontamination is possible in a non-sterile environment. Bacillus and Staphylococcus -species were identified from the samples taken during the protocol. Bacillus-species are usually isolated from soil, can tolerate harsh and low nutrient environments, and can form spores. Staphylococcus-species are part of the human skin microbiome. Microbes inside clean area are originated from personnel or surface of material transferred there. Material surface bioburden creates a contamination risk of aseptically prepared products, and thus material transfer and disinfection are critical stages during aseptic processing.