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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.