Browsing by master's degree program "Utbildningsprogrammet för provisorsexamen"

Digitalization of health care and the corona pandemic have increased availability and use of online services provided by community pharmacies. In Finland, willingness to use online pharmacy services has been studied from population approach. Less is known about the user satisfaction with the core online pharmacy services such as dispensing and medication counseling services. This study aimed to investigate satisfaction with the University Pharmacy’s online services (ya.fi) from customers’ approach. Primarily, customer satisfaction with dispensing and medication counseling services was assessed. In addition, characteristics affecting customer satisfaction were analyzed. The conceptual framework of the study was Andersen's Model of Health Services Use. The data for this study was collected by a cross-sectional survey conducted in August 2020 among University Pharmacy’s online pharmacy customers who had made a purchase during the last three months. The survey instrument consisted mainly of structured Likert-scale questions, which were used to form two sum variables: satisfaction on online dispensing services (3 variables, Cronbach's alpha 0.803) and satisfaction on online counseling services (2 variables, Cronbach's alpha 0.883). Satisfaction on online dispensing services was studied through willingness to recommend and use the services in the future. Satisfaction on online counseling services was studied through a comparison of medication counseling on an online pharmacy and a conventional pharmacy. IBM SPSS (28) -software was used for statistical analysis consisting of bivariate (Kruskal-Wallis and Mann-Whitney U tests) and multivariate (generalized linear model) analyses to identify factors affecting satisfaction with dispensing and medication counseling services. Of 15 172 invitations sent to fill out the survey, 2555 eligible responses were received (16 %). Of the respondents, 92 % had concomitantly used the services of a conventional pharmacy. . The mean of satisfaction on online dispensing services on a scale from 1 to 5 (5 being the most positive option "completely agree") was 4.3 (SD 0.8). Similarly, the mean of satisfaction on online counseling services was 3.7 (SD 0.9). According to the multivariate analyses, significant characteristics affecting satisfaction on online dispensing services were age, form of living (alone/family with children/couple), purchase of prescription or OTC medicine, frequency of internet use and previous visits to a conventional pharmacy. Characteristics affecting satisfaction on online counseling services were education, purchase of prescription medicine, use of chat information service, frequency of internet use and previous visits to a conventional University Pharmacy outlet. The services on ya.fi online pharmacy rated most important by the respondents were services about medicine availabilities (in conventional University Pharmacy outlets and during a shortage) and information about medicines (prices, Kela reimbursements and information about customers' prescriptions). Customers were satisfied with online dispensing and counseling services. Online dispensing services received a higher satisfaction rate than online counseling services. Customers who had used the chat service and purchased a prescription medicine online were more likely to assess online counseling services to be equal or better than in a conventional pharmacy. Active use of internet and purchases of medicines online were factors connected to higher satisfaction with online dispensing services. Most online pharmacy customers had also visited conventional pharmacies. The results from this study can be utilized in the development of online and other pharmacy services.

Prolyl oligopeptidase (PREP) is endopeptidase which cleaves short proline containing peptides. Abnormalities in brain PREP activity has been connected to neurodegenerative diseases. Recently it has been detected that besides its proteolytic activity PREP interacts directly with other proteins which might contribute to generation of neurodegenerative diseases. Further it has been discovered that certain small molecular PREP inhibitors are able to modify these protein-protein interactions (PPIs) and thus have a potential to alleviate the progression of neurodegenerative diseases. This has led to the development of novel second generation PREP ligands which lack the strong inhibitory activity but are potent compounds on modifying the PPIs. Thiazole structure containing PREP modulators has provided most promising class of compounds. It has been detected that these compounds mediate their effects via novel binding site on the enzyme and these effects are not connected to the inhibition of the enzymatic activity. The synthesis of these thiazole containing PREP modulators has proven to be demanding since it have involved a usage of laborious synthesis route and provided low yields. The aim of this research was to examine the synthesis of 2-(2-benzimidazol-1-yl)ethyl)- 4-methyl thiazole containing PREP modulators via previously reported synthesis route. Another aim was to design and develop a synthesis route for 2-(2-(benzimidazol-1- yl)ethyl)-5-bromo-4-methylthiazole, a molecule which serves as valuable intermediate for the lead optimization and generation of second-generation PREP modulators. A synthetic route for 2-(2-(benzimidazol-1-yl)ethyl)-5-bromo-4-methylthiazole was successfully developed. Despite that the total yield of the route remained low. When searching the reasons for the low obtained yield the chemistry behind a thiazole creating cycloaddition reaction and an aromatic halogenation was examined. This led to the discovery of a rare cationic compound which was found to be synthesized from previously undescribed starting materials.

Drug transporters and metabolizing enzymes have an important role in drug absorption in the small intestine. Food-drug interactions can affect the function of drug transporters and metabolizing enzymes in the small intestine and hence the bioavailability of drugs may change. Certain beverages have clinically relevant interactions with drugs and drinking of them should be avoided during certain drug treatments. However, possible food-drug interactions need more in vitro and in vivo studies, for example in the case of food additives which are used in the food industry increasingly, to investigate their clinical significance as inhibitors. Overall, investigating food-drug interactions is important as they might be as relevant as drug-drug interactions, especially for drugs that pass the gut wall mainly via transporters or have high presystemic metabolism. In this thesis, the inhibitor potential of 23 food additives was studied toward intestinal transporters and CYP enzymes. The food additives included sweeteners, colorants, and antioxidants. Food additives were tested against four efflux transporters with vesicle transporter assays and in OATP2B1 influx transporter with HEK293 uptake assay. The inhibition of CYP enzymes was tested in human intestinal microsomes. Six food additives were identified as possible inhibitors of BCRP, MRP2, OATP2B1, or P-gp. Two food additives were dual inhibitors. IC50 values were determined in dose-response studies for the potential inhibitors. The IC50 values were compared to the maximum expected concentration in the intestinal lumen to evaluate if the in vivo inhibition of intestinal transporters is possible. Only one food additive had a higher IC50 value than the maximum expected concentration. Eight food additives, specifically six antioxidants and two colorants, inhibited CYP-enzyme metabolism by more than 50%. Based on the results of this thesis, further studies could be performed for the identified inhibitors whose daily consumption is higher than the IC50 value. Certain food additives may inhibit CYP enzymes and the microsome assay used in this thesis is valid and could be used to study the metabolism of intestinal drug-metabolizing enzymes. However, the inhibition of transporters and CYP enzymes could be tested in cell lines, for example Caco-2 cells, to have more realistic intestinal test conditions.

Ischemic heart disease (IHD) and subsequent heart failure are caused by irreversible loss of contractile cardiomyocytes due to low oxygen supply to the heart. As the leading cause of death worldwide, IHD raises an urgent need for regenerative therapies that prevent or reverse loss of cardiomyocytes. The fetal mammalian heart grows by cardiomyocyte proliferation and utilizes glycolysis as main energy metabolism pathway, until it is introduced to increased oxygen and fatty acid supply at birth. Subsequently, cardiac energy metabolism shifts from glycolysis to β-oxidation of fatty acids and cardiomyocytes exit the mitotic cell cycle. Due to cessation of proliferation the heart can no longer regenerate after ischemic injury and responds to it by introduction of maladaptive pathological processes leading to heart failure. To gain deeper insight on the roles of cardiac metabolism pathways and hypoxia in cell cycle activation, we evaluated the effects of pharmacological metabolic modulation and oxygen supply on cardiomyocyte phenotype and hypoxia response. Furthermore, we studied the changes in the metabolic genotype of cardiomyocytes under alterations of oxygen supply. We utilized quantitative reverse transcription PCR (qRT-PCR) to evaluate the effects of hypoxia and metabolic maturation on the expression of genes involved in hypoxia signaling and metabolism of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Additionally, we investigated the effects of five metabolism-modulating compounds on cell cycle and phenotype of both metabolically matured and unmatured hiPSC-CMs, by utilizing high content analysis. We observed presence of hypoxia signaling as an increase in vascular endothelial growth factor A (VEGFA) expression following 3-hour hypoxic exposure. High expression of succinate dehydrogenase complex flavoprotein subunit A (SDHA) in hiPSC-CMs, which was downregulated at hypoxia, confirmed occurrence of oxidative metabolism induced by metabolic maturation. Surprisingly, metabolic maturation tended to increase proliferation and decrease stress response signaling of hiPSC-CMs. Introduction of the TCA cycle intermediate succinate decreased proliferation of metabolically unmatured hypoxic hiPSC-CMs by 8.2 %. Finally, inhibition of the mevalonate pathway and ketogenesis caused no alterations in hiPSC-CM phenotype or cell cycle, but introduction of the ketone body β-hydroxybutyrate tended to increase proliferation, supporting current evidence that ketogenesis plays a role in cardiomyocyte cell cycle regulation. Our observations suggest that hypoxic hiPSC-CMs can be useful in investigating gene expression and phenotype. Even so, additional methodologies are needed for in-depth evaluation of metabolic reprogramming and its effects on cardiomyocyte phenotype.

Lääketieteen kehittyessä yksilöllisen lääkehoidon tarpeeseen on kiinnitetty enemmän huomiota kuin aikaisemmin ja etenkin lapsille lääkkeiden tarkka annostelu on erityisen tärkeää. Kaupallisilla valmisteilla tarpeeksi pienet annokset eivät usein ole mahdollisia eikä tablettien puolittaminen takaa tarkkaa lääkkeiden annostelua. 3D-tulostamista on ajateltu mahdollisena vaihtoehtona ex tempore -lääkkeiden tuotantoon ja sen mahdollisuuksia on tutkittu laajalti viime vuosien aikana. Tämän tutkimuksen tavoitteena on selvittää, miten ekstruusiomenetelmällä tulostetut varfariinikalvot vertautuvat sairaala-apteekin käyttämiin varfariiniannosjauheisiin, sekä olisiko kyseistä menetelmää mahdollista hyödyntää sairaala-apteekeissa. Tutkimuksessa valmistettiin puolikiinteän aineen ekstruusiolla 0,1 mg:n, 0,5 mg:n ja 2 mg:n varfariinikalvoja, jotka kuivattiin 85 ℃:ssa valmistusprosessin nopeuttamiseksi. Kalvoja verrattiin saman vahvuisiin varfariinia sisältäviin sairaala-apteekin valmistamiin annosjauheisiin. Kalvoissa käytettiin hydroksipropyylimetyyliselluloosaa kalvonmuodostaja-aineena ja glyserolia tuomaan plastisuutta. Annosjauheet koostuivat kaupallisesta 5 mg:n Marevan-valmisteesta ja täyteaineena käytetystä laktoosista. Molemmista lääkevalmisteista mitattiin liukenemisnopeus ja annosyksiköiden yhdenmukaisuus. Molempien valmisteiden toimivuus nenä-mahaletkussa tutkittiin myös, sillä kalvojen on tärkeää soveltua erilaisille potilasryhmille. Kalvot olivat kovia, mikä aiheutti niiden hitaan liukenemisen. Puolikiinteän aineen valmistus ja tulostuksen toteuttaminen tavoitteiden mukaisesti osoittautui oletettua vaikeammaksi. Kalvoissa mitattiin annosjauheita tasaisempi lääkeainepitoisuus. Molempien lääkevalmisteiden kohdalla huomattiin, että kaikki varfariini ei pääse nenä-mahaletkujen läpi. Tärkein huomio oli, että hyvin yksinkertaisella formulaatiolla on mahdollista tuottaa lupaavia lääkevalmisteita. Tämä tutkimus esittelee syitä, joiden vuoksi 3D-tulostusta on hyvä tutkia mahdollisena ex tempore -valmistuksen menetelmänä.

Etätyö yleistyi maaliskuussa 2020 äkillisesti COVID-19 pandemian seurauksena maailman terveysjärjestö WHO:n suosituksesta. Etätyö on ollut ennen koronapandemiaa harvinaista lääketeollisuudessa, joten etätyötä lääketeollisuudessa on tutkittu hyvin vähän. Etätyön on arvioitu jäävän pysyväksi ratkaisuksi, joten on ajankohtaista tutkia etätyön soveltuvuutta ja tehokkuutta lääketeollisuudessa. Etätyöntekijöiden tuottavuus kasvaa yleensä huomattavasti. Työpaikalla koetaan jatkuvasti keskeytyksiä, melua ja muita häiriötekijöitä, joiden lisäksi työmatkat kuormittavat työntekijöitä. Etätyöntekijät säästyvät suurimmalta osalta näistä ongelmista, jolloin suurempi osa heidän työpäivästään kuluu varsinaiseen työntekoon. Valtaosa etätyöntekijöistä tekee etätyötä osan työajastaan. Tutkimuksen tavoitteena oli selvittää kokemuksia etätyöstä, etätyön soveltuvuutta ja etätyön tehokkuuteen vaikuttavia tekijöitä lääketeollisuudessa. Tutkimus on toteutettu Orion Oyj:n Suomen toimipisteissä. Tutkimuksen toteuttamistapa oli kvantitatiivisen ja kvalitatiivisen kyselytutkimuksen yhdistelmä. Yhdistämällä kvantitatiivisia ja kvalitatiivisia kysymyksiä pyrittiin saamaan tarkempia tietoja kuin pelkällä kvantitatiivisella tutkimuksella voitaisiin saada. Kysely oli avoinna 15.11.–26.11.2021. Vastausprosentiksi saatiin 34,9 %. Etätyön merkittävimmiksi hyödyiksi havaittiin työ- ja vapaa-ajan joustavampi yhteensovittaminen ja se, että etätyössä keskittyminen on parempaa. Kommunikaation koetaan onnistuvan hyvin etätyössä, mutta kasvokkain tapahtuvaa kommunikaatiota pidetään myös tärkeänä. Esimerkiksi kehitys- ja ideointipalaverit olisi hyvä järjestää mahdollisuuksien mukaan kasvokkain. Lisäksi hiljaisen tiedon siirtyminen on vähäisempää etätyössä. Etätyö soveltuu hyvin tutkimus- ja tuotekehitystyöhön, eikä sen koeta heikentävän merkittävästi kykyä innovoida. Tulosten perusteella etätyötä haluttaisiin tehdä enemmän kuin 40 % työajasta ja etätyötä pidetään tehokkaana työskentelytapana. Kyselyssä ei selvitetty, minkälaisia etätyömääriä vastaajat ovat tehneet vahvan etätyösuosituksen aikana. Osa vastaajista on saattanut olla muita enemmän lähitöissä, mikä voi vaikuttaa tuloksiin. Saadut tulokset olivat kuitenkin samansuuntaisia kuin aikaisemmissa tutkimuksissa. Suurin osa tähän kyselyyn osallistuneista oli erittäin kokeneita ja työnsä hyvin osaavia työntekijöitä, mikä voi lisätä etätyömyönteisyyttä tuloksissa.

Microcrystalline cellulose (MCC) is a purified, partially depolymerized cellulose, which is obtained by treating α-cellulose with mineral acids. Ever since the first microcrystalline cellulose was commercialized, different grades of microcrystalline cellulose have widely been used in the manufacture of solid dosage forms, such as tablets. MCC obtained from different sources will exhibit different physico-chemical properties, including moisture content, degree of polymerization, crystallinity, and particle morphology. In wet granulation, microcrystalline cellulose can be used as a filler, binder, and disintegrant. Recently, Aalto University has introduced a novel microcrystalline cellulose obtained from renewable raw materials by an integrated process, which has a short retention time, low energy and chemical consumption. However, very few studies have evaluated the use of AaltoCellTM as an excipient in solid dosage forms. The objective of this study was to evaluate the filler properties of three grades of AaltoCellTM to prepare paracetamol tablets with 50% (w/w) drug load and compare AaltoCellTM with a commercial microcrystalline cellulose, Vivapur 101. Due to the poor flowability of paracetamol and the experimental microcrystalline celluloses, it is challenging to direct compress tablets from paracetamol and microcrystalline mixtures. Thus, the powder mixtures were granulated by high-shear wet granulation method to improve the flowability. After the granulation, the formulations were characterized for particle size distribution, morphology and powder flow. Carr’s index Hausner ratio and angle of repose were calculated to evaluate the flowability of the formulations. In addition, an image-based analysis of powder flow was performed. A rotary tablet press equipped with single punches of 9 mm diameter was used to compress tablets. To evaluate the quality of tablets, European Pharmacopoeia tests of friability, disintegration, uniformity of mass, uniformity of content and dissolution were conducted. The AaltoCellTM A and Vivapur 101 formulations had the smallest particle size, whereas the AaltoCellTM B had the largest particle size. According to Carr’s index and Hausner ratio, the flowability of AaltoCellTM powders and Vivapur 101 varied from poor to very, very poor. After the granulation, the flowability of AaltoCellTM B and AaltoCellTM C were classified as good, while AaltoCellTM A and Vivapur 101 formulations had fair flowability. However, the results were conflicting with the flowability index values obtained in the image-based analysis. According to the results, the AaltoCellTM tablets complied with all criteria of European Pharmacopoeia and were comparable with Vivapur 101 tablets. The average tablet weight deviated ± 3.2% from the target weight. The variations in weight and drug content were small, as indicated by low RSD values. The disintegration time of the AaltoCellTM tablets was between 1-8.5 minutes. In addition, the AaltoCellTM tablets had fast dissolution with 78-84% of paracetamol released within 1 minute. Overall, AaltoCellTM is a promising excipient for use as a filler in tablets. In further studies, characterizing the powder properties, such as morphology, surface properties and hygroscopicity, would provide a better understanding of the properties of AaltoCellTM.

Parkinson’s disease is a progressive neurodegenerative disorder which is commonly treated with Levodopa (L-dopa) and Dopa Decarboxylase (DDC)/ Catechol-O-methyltransferase (COMT) inhibitors. The main problem with this treatment is the intestinal conversion of L-dopa to dopamine despite DDC and COMT inhibition which probably occurs by the Tyrosine Decarboxylase (TyrDC) of intestinal bacteria. This study aims to find new inhibitor molecules that would have dual inhibitory effects towards both DDC and TyrDC enzymes. Currently, available DDC inhibitors cannot inhibit the bacterial TyrDC enzyme. A recently found TyrDC inhibitor (S)-α-Fluoromethyltyrosine (AFMT) is not able to inhibit the human DDC enzyme, respectively. The dual inhibition of both decarboxylases could reduce the dosing frequency and side effects related to L-dopa. In addition, the object of this study is to produce the human DDC enzyme by DNA recombinant technique as well as develop and optimize a biochemical DDC inhibition assay to study the effect of selected small molecule compounds towards inhibition of DDC and L-dopa conversion in E. faecalis model by previously developed cell-based assay. The human DDC was successfully produced in a TB medium with a yield of 1.8 mg/mL. The Km value of DDC for L-dopa was found to be 34 μM which indicates a high affinity for L-dopa. In the optimization of the DDC inhibition assay, the sample volume of 80 μL and incubation time of 3 h with detection reagent was found to give the highest fluorometric signal with sufficient robustness. In the initial screening of test compounds, 14 % of the compounds (n=59) were classified as active towards human DDC, while 31 % of the compounds were active towards L-dopa conversion in the E. faecalis model. Of those compounds, five were having dose-dependent dual inhibitory effects, but the IC50 values of them were higher compared to either carbidopa or AFMT. The most effective compounds were 8009-2501 (IC50 37 μM in E. faecalis model and 19 % inhibition at 1000 μM towards DDC enzyme) and 8012-3386 (IC50 248 μM in E. faecalis model and 37 % inhibition at 1000 μM towards DDC enzyme). However, this study confirms the possibility to find dual decarboxylase inhibitors. By optimizing the structures as well as investigating the mechanism of action, selectivity, and structure-activity relationships of the most active compounds, it is possible to find more effective dual inhibitors in the future.

Lecithin:cholesterol acyltransferase (LCAT), a key enzyme in maturating high-density lipoprotein (HDL) particles, has been targeted to promote the efficiency of reverse cholesterol transport by small molecular positive allosteric modulators (PAM) of Daiichi Sankyo. For a set of these compounds their Vmax and EC50 values and binding site in the membrane-binding domain (MBD) of LCAT have been determined. Through molecular dynamics (MD) simulations we previously found a metric that qualitatively described which compounds were active, so in this study we aimed to improve it by finding a quantitative metric. This led to the discovery of the Cα distance between CYS50 and ASN65, which correlates with this set’s Vmax values and which can be utilized to predict the Vmax values of novel compounds. Additional simulations were performed to discover whether this metric is changed by a lipid interface present, and to reveal a likely entry pathway PAMs take. As LCAT activation is likely a benign and potentially overlooked effect, we performed a virtual screen of FDA-approved compounds and secondary metabolites associated with LCAT. From secondary metabolites, a key finding was that flavonoids were overwhelmingly associated with LCAT and had a high binding potential to the MBD in docking simulations. The best binding compounds were subjected to MD simulations to discover their Vmax values using the discovered metric. This provided us with a set of compounds, which can be used to validate our in silico model in vitro. Should this model be validated, it can be used in optimising and discovering novel PAMs of LCAT, and it would bring evidence to the benefit of MD in drug discovery processes in general. Furthermore, if our discovered compounds can activate LCAT in vitro, they may be used as precursors for novel PAMs or as therapies by themselves not only for LCAT deficiencies, but perhaps for atherosclerotic cardiovascular diseases as well.

Introduction: European legislation on orphan medicinal products, Regulation (EC) No. 141/2000 of the European Parliament and of the Council, entered into force in April 2000. Although the prevalence of rare diseases is low according to legislation (less than 5/10,000), 18–30 million people in the European Union (EU) are affected by rare diseases. The introduction of orphan medicine legislation has increased the number of orphan medicines developed but the fairness of the legislation has also raised concern and criticism. The literature review of this Master ́s thesis provides an overview of rare diseases, orphan medicines and EU orphan medicine legislation. The aim of the empirical study was to investigate the evolution of orphan medicine selection during European legislation on orphan medicinal products in 2000–2022. In more detail, aims were to describe the evolution of orphan medicine selection, the approved indications for orphan medicines and the number of orphan medicines approved for children. Methods: The research material was orphan medicines that received a marketing authorisation during the EU orphan drug legislation. This material was collected from the European Commission's Community Register of orphan medicinal products and the European Commission's Community Register of not active orphan medicinal products. Qualitative document analysis was used as the research method, where information on orphan medicines were quantified. Results and conclusions: In the 10-year review of orphan medicine development, the number of new orphan medicine products approved for the market doubled, being 63 products between 2001 and 2010 and 127 products between 2011 and 2020. In the latter 10-year period of the review, the focus of approved indications for orphan medicines shifted slightly from orphan medicines developed for the treatment of cancers (36%) to orphan medicines developed for the treatment of inborn errors of metabolism or immune disorders (43%). In the 10-year reviews, the relative share of orphan medicines approved for children decreased from 55 percent in 2001– 2010 to 40 percent in 2011–2020. Based on the results of the study, the fairness and targeting of the benefits of the orphan medicine legislation should be further investigated. Orphan medicine legislation should encourage the development of medicines for rare diseases for which there is no treatment at all, and for the population most affected, in other words children.