Browsing by study line "Farmaceutisk biologi"

African medicinal plants have been used to treat symptoms of infection successfully for thousands of years. However, no antimicrobial drugs have been developed from these plants. As antibiotic resistance is increasing rapidly, these traditional African herbal medicines can be an important solution in the fight against antibiotic resistance due to their antimicrobial properties. In this research, various extracts o the leaves of Combretum adenogonium (Combretaceae) and the fruits of Piper cubeba (Piperaceae) and Xylopia aethiopica (Annonaceae) were tested for their growth inhibitory effects against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. Extracts were made with methanol, water, hexane, and chloroform. In addition, water and ethyl acetate extracts were separated from an 80 % methanol extract using solvent partition. All the studied plants are used for the treatment of infections and wounds in African traditional medicine. Water was used as extraction solvent since it is commonly used in African folk medicine. Both single solvent technique and sequential extraction were used. The antibacterial effects were screened using agar diffusion and microdilution methods. The interaction between an extract and an antibiotic was measured with a checkerboard method. Time-kill experiments were performed using microdilution and plate count methods. In this study, the chloroform extract of C. adenogonium leaves gave the best inhibitory effect of all studied plants against B. cereus (MIC 78.125 µg/ml). In general, B. cereus was the most susceptible of the selected bacteria against extracts and E. coli was the one with most resistance. Time-kill test showed that the antibacterial efficacy was fairly stable throughout the 24-hour period considered with few exceptions. According to checkerboard results, C. adenogonium chloroform extract and tetracycline appeared to inhibit each other's antibacterial activity against B. cereus. However, only one extract was studied in this study, and it is possible that C. adenogonium contains compounds that would have a potentiating effect on antimicrobials. In general, C. adenogonium extracts were effective against B. cereus. The extracts of P. Cubeba were particularly effective against S. aureus. X aethiopica extracts were equally effective for both B. cereus and S. aureus. Methanol extract X. aethiopica is the only extract studied that gave more than 90% inhibition against P. aeruginosa. Therefore, it could be concluded that X. aethiopica has the broadest activity range of the examined plants.

The life cycle of Chlamydia pneumoniae is a biphasic developmental cycle, as a obligate intracellular bacterium, it forms various morphological forms, including elementary bodies, reticulate bodies and aberrant bodies belonging to a persistent form. Due to the bacterial life cycle and the fact that chronication of C. pneumoniae infection and formation of persistent infection as well as pathogenesis is a complex problem involving multiple signaling pathways and affecting several different cells, it is useful to seek medication to influence infection from different stages of the bacterial life cycle. There are several different factors that induce persistence and thus models of persistence. Although the detection of aberrant RBs and thus aberrant bodies in C. pneumoniae infected tissues does not provide complete certainty about chronic infection, the bacterium has been linked to chronic health problems such as atherosclerotic cardiovascular disease and asthma. The aim of the study was to develop a persistence model induced by beta-lactam antibiotics, amoxicillin and penicillin G, in A549 cells by monitoring the size, shape, and number of inclusions using the IPA method and the immunofluorescence staining method for infection. In addition, the antibiotic sensitizing effect of three compounds on pulmonary chlamydial infection was studied. This effect was monitored by examining the recovery of persistent infection and by monitoring the protective effect of the compounds on beta-lactam-induced persistence. The work succeeded in finding an infection model that is well suited for studying beta-lactam persistence. Due to treatment recommendations, pulmonary chlamydial infections are practically treated with beta-lactam antibiotics. Based on the methods used, it was found that amoxicillin concentrations of 10 and 25 µg/ml and penicillin G concentrations of 100 U/ml and 250 U/ml were sufficiently effective to transfer bacteria to a state of persistence. It was found that the amoxicillin persistence model is reversible based on the increase in the size of the inclusions, especially at 25 µg/ml and quantitatively at 10 µg/ml. It was concluded that amoxicillin at a concentration of 10 µg/ml is sufficient to induce persistence in a beta-lactam antibiotic-induced persistence model. Further quantitative studies on the persistence model are needed, such as quantitative PCR based on the OmpA gene to determine more accurate dose-response relationships. Glutathione levels should also be monitored in the persistence model.

Chilit ovat Capsicum-sukuun kuuluvia yleensä korkean kapsaisiinipitoisuuden omaavia paprikalajeja. Niitä käytetään mausteena. Lisäksi chilien sisältämillä kapsaisinoideilla on todettu olevan useita farmakologisia ominaisuuksia, kuten analgeettisia ja antioksidanttisia vaikutuksia. Niiden antimikrobisia ominaisuuksia on myös hieman tutkittu, mutta tutkimuksia on vielä verrattain vähän. Tämän työn tarkoituksena oli selvittää muutamien eri chililajikkeista valmistettujen uutteiden antimikrobisia vaikutuksia Escherichia colia ja Staphylococcus aureusta vastaan. Uutteet testattiin dimetyylisulfoksidiin (DMSO) ja veteen liuotettuina. Lisäksi testattiin myös kahden puhdasaineen, kapsiaatin ja solaniinin, vaikutuksia kyseisiä bakteereita vastaan. Antimikrobiakokeet suoritettiin 96-kuoppalevyllä noudattaen aseptisia työtapoja. Testattuja chiliuutteita oli 19. Uutteita valmistettiin eri chililajikkeiden versoista (1 kpl) siemenistä (3 kpl), lehdistä (10 kpl) ja hedelmistä (5 kpl). Dimetyylisulfoksidiin liuotetut uutteet testattiin pitoisuuksilla 2,0 mg/ml ja 4,0 mg/ml. Veteen liuotetut uutteet testattiin pitoisuudella 4,0 mg/ml. Solaniini- ja kapsiaattiuutteet testattiin kahdeksalla eri pitoisuudella (0,001172–0,15 μg/ml). Tutkimuksen tuloksena on, että testatut chiliuutteet eikä solaniini- ja kapsiaattiuutteet estäneet E. colin tai S. aureuksen kasvua. DMSO:iin liuotetuista uutteista korkeimmat estoprosentit kumpaakin bakteeria vastaan saatiin nuorilla Pimento-lehdillä. Veteen liuotetuista uutteista korkein estoprosentti E. colia vastaan saatiin Dulcen versoilla (30 % esto) ja S. aureusta vastaan Dulcen hedelmillä (50 % esto). Aiemmat tutkimustulokset chilien antimikrobisista vaikutuksista ovat ristiriitaisia, joten yhteneviä johtopäätöksiä chilien vaikutuksista bakteereihin ei voida tehdä. Johtopäätöksenä voidaan todeta, että chileillä on lukuisia terveysvaikutuksia. Antimikrobisen tehon varmistamiseksi tarvittaisi kuitenkin lisää tutkimuksia. Antibioottiresistenssi on maailmanlaajuinen ongelma, koska yhä useammat bakteerit ovat resistenttejä käytetyille antibiooteille. Tulevaisuudessa onkin erittäin tärkeää löytää uusia yhdisteitä bakteerien tappamiseksi, joten tutkimuksia uusien antimikrobisten aineiden löytämiseksi tarvitaan jatkuvasti lisää

Drug metabolism is a series of enzyme catalysed processes that modify foreign compounds into a form that is more easily excreted from the body. Compounds can affect the activity of metabolizing enzymes and this may lead to toxic concentrations of a drug that is metabolized via the enzyme. With prodrugs, on the other hand, the drug might not achieve its biologically active form and therefore the treatment will not be effective. Recognizing and preventing metabolic interactions is important already in the early stages of drug discovery and development. Cytochrome P450 (CYP) enzyme inhibition is one of the major reasons for adverse drug-drug interactions (DDIs). The inhibition can be time-dependent (TDI), which means that the potency of inhibition increases over time. TDI may be reversible or irreversible, latter being more severe as new enzymes need to be produced in the body to restore the enzymatic activity. IC50 shift assay is a method that gives information of new compounds potential to cause TDI. IC50 shift assay does not show whether the TDI is reversible or irreversible, however further studies, e.g. dialysis assay, can be conducted to find it out. If the study compound is irreversibly bound to the enzyme, the enzyme activity should not recover in the dialysis. The aim of this master’s thesis was to develop a dialysis method that could determine the reversibility of the TDI observed in the IC50 shift assay. A dialysis method conducted with microsomes is described in earlier literature. Known inhibitors (both time-dependent and direct) for four CYP isoforms were studied in this work: CYP1A2 (furafylline and fluvoxamine), CYP2C9 (tienilic acid and sulphaphenazole), CYP2D6 (paroxetine and quinidine) and CYP3A4 (verapamil, azamulin and ketoconazole). IC50 shift assays were conducted to each inhibitor before the dialysis experiment. The studied compounds behaved in the dialysis assay mostly as assumed based on the literature. The workflow from IC50 shift assay to dialysis assay worked successfully and the IC50 shift data could be utilized when choosing the test concentrations for dialysis assay. Both the IC50 shift assay and dialysis assay were reproducible and the deviations between replicates and separate studies were relatively low. The method still requires some optimizing, but so far, the results are promising. In the future the dialysis method may be part of in vitro CYP inhibition studies at Orion Pharma.

The use of different methods of extended reality (xR) as a support in teaching has been under research for a long time. Although the use of various xR-technologies in other fields of healthcare, such as medical and nursing education, is already common, their use in pharmacy education is not yet well established. There is evidence that xR-technology has a positive impact for example on students’ motivation and learning outcomes. On the other hand, there are limiting factors that inhibit the technology becoming widespread, such as costs as well as a lack of knowledge about the technology usability. The aim of the study was to investigate usability and advantages of the augmented reality (AR) in a laboratory course as an educational supportive tool by using AR-glasses. The aim was also to investigate the learning outcomes of the students who participated in the study in three different phases: before carrying out the laboratory work (pre), immediately after the laboratory work (post) and in the course exam (delayed). Furthermore, the motivation of the students to use new technology in their studies was studied. The research was done in a collaboration with the Centre for University Teaching and Learning (HYPE) and with Sciar Company Oy. The researchers of HYPE were responsible for the pedagogical point of view, whilst the experts from the Faculty of Pharmacy were responsible for the study measurements of laboratory work related content knowledge. The research was implemented in two laboratory courses in Bachelor of Science level pharmacy studies: Medicinal product II and Pharmaceutical biology and asepsis in the fall of 2020. The students (n=18) prepared eye drops by using AR-glasses in the Medicinal product II -course and reference group (n=14) without AR-glasses. In the course of Pharmaceutical biology and asepsis, students (n=7) used AR-glasses to study the microbiological purity of the eye drops by utilizing membrane filtration method in cleanroom and reference group (n=9) without AR-glasses. ln addition, a serial dilution method was performed on a 96-well plate using an AR mobile application. The effect on learning outcomes was evaluated by using six open-ended questions measuring the understanding of content knowledge underlying the laboratory work, that were answered by the students at three different stages of the study (pre, post, delayed). To measure the usability of the AR equipment, a five-point Likert scale questionnaire studied the experimental groups students’ opinions on whether the AR mobile application could provide sufficient guidance and feedback while performing the laboratory work. In an open question, the students had the possibility to comment on the overall user experience of the AR mobile application. There were no statistically significant differences in learning outcomes between the AR-group and the reference group in both laboratory courses. The results showed indicative differences in short-term and long-term learning, with the AR-group achieving better learning outcomes in the short-term and the reference group in the long-term. In the course of Pharmaceutical biology and asepsis, the learning outcomes were the opposite. Students’ were found to be receptivity to the new technology that together with motivation supports positively the learning process. The use of AR-hardware increased certainty and reduced nervousness about the use of AR technology. As a conclusion, the study could not demonstrate the benefit of AR-technology in student learning outcomes. The study was limited by the small sample size. However, further studies are encouraged due to students’ positive attitudes and motivation towards AR technology. Regarding further studies, it is important to take into consideration the different backgrounds and learning methods of students. Thereby, the effects of xR-technologies on learning outcomes can be assessed as objectively as possible.

Diseases caused by foodborne pathogens are a global threat, which is why new bioactive compounds are expected in the food industry. The purpose of this work was to investigate the antimicrobial effects of three different plants, blackcurrant (Ribes nigrum), rhubarb (Rheum spp.), and Scots pine (Pinus sylvestris), against seven pathogenic bacteria. Bioactivity of these plants has been previously shown, but results have varied widely depending for example on the plant part, extraction solvent and pathogen. The plant samples were extracted with 30 % or 80 % ethanol-water solution. There was a total of 12 extracts: rhubarb petiole (dried at 45 °C or lyophilized), rhubarb root (dried at 50 °C), blackcurrant berry (dried at 45 °C or lyophilized) and lyophilized juice of Scots pine needles. Extracts were dissolved in dimethyl sulfoxide and bioactivity screening of the extracts was determined at a concentration of 1,0 mg/ml, after which the active extracts were subjected to minimum inhibitory concentration (MIC) determination (n=2-3) at eight concentrations (0,0625-4,0 mg/ml). Antimicrobial experiments were performed on a 96-well plate following Clinical and Laboratory Standards Institute guidelines. Bioactivity was determined based on absorbance measurements and visual inspection. The extract of rhubarb root showed most potential against tested bacteria. The lowest MIC values (0,25 mg/ml and 0,50 mg/ml) were obtained with rhubarb root extracts (extracted with 80% or 30 % ethanol-water solution) against Staphylococcus aureus and Bacillus cereus and 1,0 mg/ml against Listeria monocytogenes. Based on this study rhubarb root extract could be a potential natural antimicrobial against foodborne pathogens.

Antibiotic resistance is a global crisis causing increasing number of infections that cannot be treated with conventional antibiotics. The main reasons for the resistance crisis include overuse and misuse of antibiotics, use in agriculture, and decreased interest of pharmaceutical companies to discover and develop new antibiotics. Apart from mortality, antibiotic resistance causes large economical costs due to longer hospitalizations and more expensive treatments. Bacteria can acquire resistance via multiple pathways. Main path for spread of resistance in bacterial populations is horizontal gene transfer (HGT) in which bacteria receive genetic material that contain resistance genes. Bacteria can acquire new resistance traits via mutations in their genome. The emergence of resistance is a natural feature of bacteria and therefore many bacteria and bacteria can quickly acquire resistance towards novel antibiotics. The resistance properties of potential new antibiotics should be studied already during drug discovery and development. This study determined resistance properties of diazaborine compounds, which have been shown to have antibacterial activity especially against Gram-negative bacteria. The studied properties were mutant prevention concentration (MPC) and spontaneous mutation frequency. MPC measures the antimicrobial compound concentrations in which single-step resistant mutants arise. MPC values can be compared to minimum inhibitory concentration (MIC) values to determine range of mutant selection window (MSW) in which single-step resistant mutants are selectively amplified. Spontaneous mutation frequency is a feature for bacteria in presence of antimicrobials. Spontaneous mutation frequencies demonstrate the proportion of emerged resistant mutants from a bacterial population in each antimicrobial concentration. Four diazaborine compounds were studied with Escherichia coli ATCC25922 and the results were compared with ciprofloxacin. Ciprofloxacin had the lowest MPC at 16xMIC, diazaborine compounds 1, 2 and 3 at 32xMIC and diazaborine compound 4 at 64xMIC. Spontaneous mutation frequency of E. coli was on a normal level with each compound.

Kasvavan antibioottiresistenssin vuoksi terveydenhuollossa tarvitaan uusia antibiootteja ja antibioottien apuaineita. Tästä syystä tässä työssä tutkittiin Combretaceae- ja Annonaceae-heimoihin kuuluvien tansanialaisten lääkekasvien antibakteerisia vaikutuksia. Kansanlääketieteessä näitä kasveja on perinteisesti käytetty mm. bakteerien aiheuttamien sairauksien ja oireiden hoitoon, mikä antaa viitteitä siitä, että ne sisältävät antibakteerisia yhdisteitä. Tutkimuksen tarkoituksena oli pyrkiä löytämään raakauutteita ja neste-nesteuutolla saatuja fraktioita, joilla on mahdollisimman hyvät estovaikutukset bakteerien kasvuun. Lisäksi oli tarkoitus selvittää Annonaceae-heimon lajien sisältämiä yhdisteitä, mikä voi osaltaan auttaa löytämään uusia antibiootteja. Antibakteerisia tutkimuksia tehtiin Combretaceae- ja Annonaceae-heimoihin kuuluvien Combretum-, Terminalia-, Friesodielsia- ja Hexalobus-sukujen lajien uutteille ja fraktioille käyttäen agardiffuusio- ja mikrodiluutiomenetelmiä. Yhteensä 45 eri uutteen ja fraktion estovaikutuksia tutkittiin ruokamyrkytyksiä aiheuttavien Bacillus cereus - ja Salmonella enterica -bakteerien kasvuun. Lisäksi tutkittiin Annonaceae-heimoon kuuluvien Friesodielsia obovata - ja Hexalobus monopetalus -lajien uutteiden ja fraktioiden sisältämiä yhdisteitä käyttäen HPLC-DAD - ja UHPLC/Q-TOF-MS -menetelmiä. Tutkimustulosten perusteella useilla Combretaceae- ja Annonaceae -heimoihin kuuluvilla lajeilla on antibakteerisia vaikutuksia grampositiivista B. cereus -bakteeria vastaan, mutta ei niinkään gramnegatiivista S. enterica -bakteeria vastaan. Aiemmissakin tutkimuksissa on usein, ei kuitenkaan aina, saatu parempia estovaikutuksia grampositiivisten kuin gramnegatiivisten bakteerien kasvuun. Tässä tutkimuksessa parhaimmat tulokset (MIC = 156 µg/ml) saatiin mikrodiluutiomenetelmällä B. cereus -bakteerin kasvun estoon Combretum fragrans -lajin lehtien kuumalla Soxhlet-metanoliuutteella ja Friesodielsia obovata -lajin lehtien metanoliuutteen veteen liukenemattomalla fraktiolla. Tarvitaan kuitenkin vielä paljon lisätutkimuksia, varsinkin H. monopetalus - ja F. obovata -lajien kohdalla, jotta voidaan selvittää, voidaanko tutkituista kasviuutteista ja niiden fraktioista eristää mahdollisia uusia antibiootteja tai antibioottien apuaineita ihmisten ja eläinten infektioiden lääkintään. Annonaceae-heimon lajeista löydettiin samantapaisia yhdisteitä kuin on löydetty aiemmissa tutkimuksissa, mutta 6,8-dimetyyli-monohydroksi-pinosembriiniä karakterisoitiin ensimmäistä kertaa F. obovata -lajista. F. obovata -lajin lehdistä tunnistettiin ensimmäistä kertaa (-)-krotepoksidi ja krotepoksidin johdannaisia. Lisäksi H. monopetalus -lajin juuresta karakterisoitiin ensimmäistä kertaa 3-(2',3'-dihydroksi-3'-metyylibutyyli)-5-(3''-metyylikrotonyyli)indolia, 3-(1,3-dihydroksi-3-metyylibut-2-yyli)-6-(2-hydroksi-3-metyyli-3-butenyyli)indolia sekä heksalobiini C:tä ja D:tä.