Browsing by master's degree program "Master 's Programme in Pharmacy"

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.

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by the death of nigrostriatal dopaminergic neurons and formation of intraneuronal protein aggregates called Lewy bodies and Lewy neurites. These inclusions consist of a protein called α-synuclein (aSyn) but also of other proteins, lipids and cell organelles. Progressive cell death leads to nonmotor and motor symptoms. Current therapies for PD are symptomatic and do not modify the disease progression. Therefore, there is a need for the development of therapies attenuating the neurodegeneration. The pre-formed fibrils (PFF) model enables studying of aSyn aggregation and mechanisms behind inclusion formation. The PFF model is based on the exogenous aSyn fibrils’ tendency to result in formation of Lewy body -like inclusions when added in cell culture or in animals. Primary neuronal cultures of mice and rats have typically been used to model aSyn aggregation in vitro with the PFF model. Primary neuronal cultures provide practicality and are able to depict relevant features of dopaminergic neurons. To gain insight about the composition of E13.5 primary embryonic mouse midbrain culture and to enable adaptation of an existing protocol to study other cell types, this study identified and quantified several relevant cellular phenotypes in the micro island culture. The cells were fixed on day in vitro (DIV) 8 or DIV 22 and analysis was conducted using fluorescent immunocytochemistry combined with automated image analysis software, CellProfiler. On DIV 8, tyrosine hydroxylase -positive dopaminergic neurons represented 5 % of the total cells in the culture. Neuronal nuclear antigen -positive neurons resulted representing 30 % of the total cells. Gabaergic neurons were identified to be abundant in the culture and certain dopaminergic neurons were identified as immunoreactive for GABA. Choline acetyltransferase -positive cholinergic neurons were also identified to be present in the culture. The number of oligodendrocyte precursors (OPCs) was observed to be significantly smaller than the number of dopaminergic neurons. OPCs represented around 1 % of the culture on DIV 8. Glutaminergic neurons, parvalbumin-positive interneurons, microglia or astrocytes were not identified in the culture on DIV 8. The number of astrocytes was observed to increase as the incubation time was prolonged to DIV 22. Overall these findings provide valuable insights of the composition of cell phenotypes in E13.5 mouse midbrain culture. The results also provide additional validation for suitability of the original protocol to robustly produce midbrain dopaminergic cultures with minimal number of glial cells. Understanding more about the relevance and interplay of different cell phenotypes in PD pathophysiology can provide valuable insight for the development of potential therapeutic strategies.

Introduction: When people age, the composition of sleep changes and sleep becomes more sensitive to external disturbances, making insomnia also more common. Medication is not the first-line treatment option for insomnia. Benzodiazepines or benzodiazepine receptor agonists for the treatment of insomnia have been in the focus of past studies. The content of the dosing instructions for the supplied medicines has not been studied. The dosing instructions should provide clear instructions on how to dose the medicine prescribed to the patient. The aim of this study was to investigate the content and quality of dosing instructions prescribed for the treatment of insomnia for Finns aged ≥75 years in 2020 based on the prospective reimbursement register data by the Social Insurance Institution of Finland (Kela). Materials and methods: The reimbursed purchases of all medicines by persons aged ≥75 years from 1.1.2020 to 31.12.2020 were selected by ATC code from the medicines data according to the Insomnia: Current Care guidelines. The data was gathered from Kela’s dispensations reimbursed under the National Health Insurance scheme. The dataset consisted of 1,080,843 delivery lines, which were screened, and 328,285 lines were included in the analyses. Dosage instructions were reviewed according to the following predetermined five categories: frequency of use, dose, timing, warnings or remarks, and inappropriate instructions. In addition, 1000 dosing instructions were randomly derived to study the phrasing and appropriateness of the dosing instructions in more detail. Results: In 2020, an average of 3.8 reimbursed hypnotic drug deliveries were made per elderly person. Of the deliveries, 68% were for women. 52% of drug deliveries were partially made later and not by original prescription. In the hypnotic dataset, the three most administered drugs were zopiclone (41%), mirtazapine (34%) and zolpidem (12%). The dosage was prescribed in 98–99% of the dosage instructions. Dosing schedule was reported in 83% of dosing instructions and regularity of use was reported in 57% of them. Only 3-6% of the dosing instructions had comments or warnings. 1–2% of the dosing instructions were vague. The duration or regularity of use was clearly indicated in 5% of the dosing instructions. Only 0.1% of dosage instructions contained instructions for discontinuation or reduction. Discussion: The dose and timing of administration were well reported, but the frequency of use was reported in only about half of the dosing instructions. Only few dosing instructions contained remarks or warnings even though hypnotics are at risk for the elderly. Among the three most administered drugs for the treatment of insomnia were two benzodiazepine receptor agonists, zopiclone and zolpidem. However, they may not be suitable for the elderly according to Beers criteria and their use should be avoided. During 2020, an average of four drug deliveries were made per elderly person for the treatment of insomnia, which may indicate prolonged hypnotics use. In addition, more than half of the deliveries were partially made later and not by original prescription. Thus, several drug packages are prescribed for prescriptions, although the drug-based treatment of insomnia should only be short-lived. Conclusions: There are significant deficiencies in the contents and quality in the dosing instructions for drugs delivered to the elderly for insomnia. Minimum information on dose, timing and duration of use was not found in all dosing instructions in this study. Understandable dosing instructions and the reduction in the amount of medication in the prescription could have a further effect on reducing the long-term use of hypnotics, also increasing the safety of medicine use in the elderly.

Medication safety is an important target of development in health and social services systems internationally. Medication errors are one of the biggest risk factors in medication safety. Majority of the medication incidents could be avoided by improving the medication treatment process. Patient safety incident reporting systems enable health and social services to collect systematic data from risk factors within the medication treatment process. This study was conducted as a retrospective registry-based study where medication incidents that occurred in health and social care units reported by community pharmacies to the incident reporting system HaiPro from 21st of September 2021 to 31st of October 2022 were analysed. Cases that did not meet the criteria for this study (n=55) were removed from the original data (n=3841). If needed, the nature and type of the reported error were corrected. A descriptive quantitative analysis was conducted for the final data (n=3786) using Microsoft Excel. The number, natures, types, observers, and prescription types of medication errors were investigated from the data. In addition, the most common groups of medicinal substance and high risk medicines were identified. A qualitive content analysis was performed to near miss cases involving high-risk medications (n=446) using the Atlas.ti program. Interventions, measures following the interventions and risks prevented by the measures were identified from the open description in the incident reports. The qualitative analysis was performed as an abductive content analysis. Of the medication errors included in the study (n=3786) 91% were detected by community pharmacies and the majority (68%) of the reported incidents were near misses. Most (96%) of the safety incidents (n=3786) were associated with the patient’s medication treatment and had occurred mostly during the prescribing process (92%). As a result from the prescribing errors, patients were most commonly prescribed wrong dose or strength of the medicine (26%) or the prescription lacked SIC marking (26%). High-risk medications occurred in 16% (n=591) of the incidents (n=3786). Most frequently detected high-risk medications were opioids (35 %). Three quarters (76 %) of safety incidents associated with high-risk medications were near misses (n=446). The majority (92 %) of interventions (n=471) made to prevent safety incidents associated with high-risk medications were made by community pharmacies. The most frequent intervention was community pharmacies contacting the doctor. Based on the HaiPro incident reports made about medication errors in health and social care units reported by community pharmacies, it can be concluded that community pharmacies are a central barrier in primary care medication treatment process. Community pharmacies detect and report medication errors that have occurred in other health and social care units. Safety incidents reported by pharmacies systematically accumulate important information that can be used in the development of medication safety in primary care at a unit, wellbeing services county and national levels.

Operations of pharmaceutical supply chain and medication management practices will be evaluated as part of the ongoing social and health services reform in Finland. One of the goals is to develop digital medication management tools and services to meet the needs of both healthcare professionals and medicine users. The aim of this study was to examine population's willingness to use on new digital services by community pharmacies to promote rational pharmacotherapy and to support cooperation between those involved in the medication use process. The material for this study consisted of the national population survey conducted in 2020 for the VN TEAS report “Activation of price competition for pharmaceutical products and the population's expectations for pharmacy operations” (online survey for 18–79-year-old adults, n = 1650). The survey respondents represented well the target population expect those with higher educational level were over-represented. The current study focused on questions related to digital medication management services provided by community pharmacies, which were divided into the following 4 topics to form sum variables: 1) purchasing and dispensing process of medicines (4 items), 2) customer`s communication with pharmacy and health care personnel (2 items), 3) pharmacy and healthcare personnel`s communication with the customer (4 items) and 4) support services for medication self-management (12 items). The associations of the background variables to the sum variables were calculated using cross-tabulation and the Chi-Square test. Frequencies and percentages were used to present. The majority (85–90%) of the respondents were in favor of the possibility of sending messages electronically between the customer, the community pharmacy and other healthcare personnel by using a shared communication channel, such as My Kanta to update medication information online. Multimorbidity, medication use, and higher medication costs increased the respondent’s positive attitude towards the electronic communication channel. Three-quarters (76%) of respondents were willing to use electronic medicine purchasing and dispensing services. Younger respondents (18–34 years) were more interested in these services than older ones. The electronic medication self-management support services had more discrete opinions among respondents. More than half of the respondents indicated their strong willingness to use at least one of the medication self-management support online services listed in the survey instrument. Those aged 18–34 years (69%), those with higher education (62%), those living in the Province of Southern Finland (60%) and those living in the Helsinki Metropolitan Area (67%) were more positive than others. Of the respondents who opted for pharmacy's remote online services, 55% were willing to seek advice for reconciling their medication list. According to the survey, Finnish adults are willing to use new electronic services by community pharmacies. In particular, they were willing to use a shared electronic communication channel between the customer, the pharmacy and other healthcare personnel, such as MyKanta to update information related to their medication. The willingness to have support self-care support for medication self-management primarily from the pharmacy's online services was lower than the willingness to use online purchasing and dispensing services. Of the remote medication self-management services medication reconciliation had the highest demand. Future research should focus on enhancing use of electronic medication self-management services provided by community pharmacies. Further research should also be targeted to understand medicine user needs for support as it may vary between patient groups, requiring segmentation of services.

Poorly water-soluble drugs are challenging to formulate as solid oral dosage forms because of their inadequate solubility in the gastro-intestinal tract. Amorphous solid dispersions (ASDs) are a proven method of increasing the oral bioavailability of poorly water-soluble drugs through drug supersaturation. Downstream processing of ASDs into oral tablets has gained academic interest in recent years. However, minitablets, which are tablets smaller than 4 mm in size, have not received the same level of attention. Minitablets have been cited as a promising dosage form for children, the elderly and in veterinary use because of their good compliance, flexible dosing, and ease of swallowing. In this work, 15 different blends of microcrystalline cellulose and lactose have been characterized for their suitability in the formulation of an ASD of spray-dried poorly soluble indomethacin in PVP K 29-32 or HPMCAS MF as minitablets. Minitablets were compressed at the compression forces ~1000 N and ~1500 N. The flowability of the blends were evaluated based on the Carr’s indices, Hausner ratios and angles of repose. From the most promising blends, 3.0 mm placebo minitablets were manufactured. A mixing test using colored beetroot powder was used to determine the optimal mixing time. The finished tablets were tested for their uniformity of mass, crushing strength, height, and disintegration. Based on their Carr’s indices and Hausner ratios, Vivapur 105, Vivapur 200, Pharmatose 200M and Pharmatose 80M had the best flowabilities. Placebo minitablets were successfully manufactured from blends of these excipients except for the 1:1 ratio of Vivapur 105/Pharmatose 80M. The mixing test indicated that the optimal mixing time is 20 to 25 minutes. The mass variation for all placebo batches except the 1:3 ratio of Vivapur 105/Pharmatose 80M was less than 10 percent from the average mass and most batches therefore fulfilled the uniformity of mass requirement of the European Pharmacopoeia. For five of the batches, the variation was within 2.80 percent. The average crushing strengths were between 32.4 N and 79.7 N and increased with increasing compression force. All batches of placebo minitablets disintegrated within 6 to 19 seconds on average except the 1:3 ratio of Vivapur 105/Pharmatose 80M which took 90 seconds to disintegrate. Minitablets filled in capsules disintegrated within 124 to 167 seconds on average except for the previously mentioned slower disintegrating batch which disintegrated in 477 seconds. All placebo minitablets, individual or loaded into capsules disintegrated within 15 minutes thereby fulfilling the requirement of the European Pharmacopeia. When considering the results obtained for placebo minitablets, the 3:1 ratio blend of Vivapur 200/Pharmatose 200M with 0.5 % (w/w) magnesium stearate was found to be the most promising candidate for ASD formulation. This formulation was subsequently used as the basis for the manufacture of 3.0 mm minitablets containing 6.22 % (w/w) of a spray-dried dispersion of indomethacin and PVP K 29-32. Except for one outlier, the mass variation of these minitablets fell within 2.37 % of the average mass, thereby fulfilling the requirement of the European Pharmacopoeia. Single indomethacin-PVP minitablets disintegrated within 6 minutes and 38 seconds, and capsules containing twelve minitablets disintegrated within 10 minutes and 37 seconds, which also is accordance with the pharmacopoeia. At 80.3 to 80.4 N the crushing strength was at the upper end of the targeted range, but still adequate. Thus, the formulation developed in this study appears promising for the manufacture of minitablets containing 6.22 % of an amorphous indomethacin-PVP dispersion. This study demonstrated that minitablets could be manufactured from a spray-dried solid dispersion despite its poor flowability.

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

Viral promoters are an essential part of a normally functioning virus. Their main task is to drive the transcription of genes which govern hijacking of cell function and replication of viral particles. In addition to supporting normal function of a virus, they can be used to drive the transcription of transgenes which can be used in different therapies. In oncolytic therapies, transgenes can be used to prime the host system against neoplasms which has been shown to generate long term anti-tumour immunity. Human adenoviruses (Ad) are commonly used as a platform for oncolytic virotherapies. Human Ad’s replicate poorly in mouse tumour cell lines, yet some promoters, which are included in the viral constructs to drive the transcription of beneficial transgenes, are able to function. Currently it is unknown whether E3, the native promoter of adenovirus 5 of the E3 region, is capable of functioning in murine cell lines. In this thesis we study whether human cytomegalovirus promoter (CMV) and E3 differ in their efficacy to drive the transcription of the mOX40L and mCD40L transgenes. In the experimental part of this thesis, we compared the efficacies of two viral promoters, AdE3 and AdCVM, in transcribing mOX40Land mCD40L in vitro. Efficacy of transcription was assessed through immunofluorescence and flow cytometry in human and murine cell lines. Furthermore, the effects of promoters on viral infection, killing and replication were evaluated in burst assay and the colorimetric MTS proliferation assay. MTS and burst assay were conducted to confirm if viral infection, killing and replication occurs in human and murine cell lines. Both AdE3 and AdCMV were able to infect and kill human cell lines and cell viability decreased in correlation to the number of viral particles used. In murine cell lines, no decrease in cell viability was detected in the 4T1 cell line. In burst assay, viral replication was observed for both AdE3 and AdCMV in the human MDA-MB-436 cell line. In murine CT26 cell line, no replication was observed for AdE3 or AdCMV constructs. Immunofluorescence assay was performed to visualize transgene expression and localization. Results indicated that mOX40L was localized on cell surface while mCD40L was detected both outside and inside of the cytosolic compartment. Flow cytometry results revealed that both AdE3 and AdCMV constructs are capable of efficiently transcribing mOX40L in human cell lines. In the flow cytometry results for AdE3, two large cell populations with different fluorescence intensities were detected. AdCMV lacked this feature which is postulated to be due to higher lytic activity of the viral construct. In murine cell lines, HCMV could produce mOX40L, but production in murine cell lines was severely attenuated compared to human cell lines. mOX40L produced by the AdE3 construct did not differ from the baseline and was deemed incapable of producing mOX40L in murine cell lines. For the purpose of studying novel virotherapeutics the results of this thesis would indicate that human CMV can be used to drive expression of transgenes in murine cell lines. Despite this, it is preferable to use host specific viruses and promoter sequences for a better translation between mice and humans. Viruksen promoottorit ovat keskeisessä osassa toimintakykyisessä viruksessa. Virus promoottorin päätarkoituksena on geenien transkriptio, mitkä vastaavat solun keskeisten toimintojen kaappaamisesta ja virus partikkeleiden replikaatiosta. Näiden toimintojen lisäksi promoottoreita voidaan käyttää transgeenien transkriptiossa, mitä voidaan hyödyntää sairauksien hoidossa. Onkolyyttisissä terapioissa transgeenejä voidaan käyttää virittämään kehon immuunipuolustus taistelemaan kasvainkudosta vastaan. Ihmisen adenovirusta käytetään usein onkolyyttisten viroterapioiden alustana. Ihmisen adenovirus (Ad) replikoituu hyvin heikosti hiiren syöpäsoluissa, mutta osa adenovirukseen sisälletyistä eksogeenisistä promoottoreista, joita käytetään terapeuttisten transgeenien transkription ajamiseen, kykenee toimimaan ja tuottamaan haluttua proteiinia. Tällä hetkellä ei tiedetä, kykeneekö E3, joka on adenoviruksen E3 lokuksen promoottori, toimimaan hiiren solulinjoissa. Tässä tutkielmassa selvitämme ihmisen sytomegalovirus promoottorin (CMV) ja E3 eroa niiden tehossa ajaa mOX40L ja mCD40L transgeenien transkriptiota. Kokeellisessa osuudessa vertailimme kahden virus promoottorin, E3 ja CMV, eroa niiden tehossa ajaa mCD40L ja mOX40L transkriptiota in vitro. Transkription tehoa tutkittiin immunofluoresenssin ja virtaussytometrian avulla ihmisen ja hiiren syöpäsolulinjoissa. Tämän lisäksi promoottorien vaikutusta virus infektioon, replikaatioon ja kykyyn tappaa soluja arvioitiin burst kokeella ja kolorimetrisellä MTS menetelmällä. MTS ja burst kokeiden avulla varmistettiin AdE3 ja AdCMV virusten kyky infektoida, tappaa ja replikoitua ihmisen ja hiiren syöpäsolulinjoissa. Sekä Ad3 ja AdCMV todettiin kykenevän infektoimaan ja tappamaan ihmissyöpäsoluja ja solujen viabiliteetin lasku korreloi virus partikkeleiden määrän kanssa. Hiiren 4T1 syöpäsoluissa ei todettu solujen viabiliteetin laskevan. Burst kokeessa havaitsimme sekä AdE3 että AdCMV kykenevän replikoitumaan ihmisen MDA-MB-436 solulinjassa. Hiiren CT26 solulinjassa kummankaan viruksen ei havaittu kykenevän replikoitumaan. Immunofluoresenssi kokeessa visualisoimme transgeenien ilmentymisen ja paikantumisen. Tulokset osoittivat, että mOX40L paikantui solun pinnalle. mCD40L havaittiin puolestaan sekä solun ulkopuolella että sytosolissa. Virtaussytometria kokeen tulokset osoittivat, että sekä AdE3 ja AdCMV pystyivät tehokkaasti ilmentämään mOX40L ihmisen solulinjoissa. AdE3 virtausytometria tuloksissa löydettiin kaksi solupopulaatiota, joilla oli toisistaan poikkeavat fluoresenssi intensiteetit. Tätä ilmiötä ei havaittu AdCMV:lla infektoiduilla soluilla, mikä saattoi johtua korkeammasta lyyttisestä aktiivisuudesta. Hiirisolulinjoissa CMV kykeni ilmentämään mOX40L, mutta transkription teho oli selvästi alhaisempi verrattuna ihmissolulinjoihin. E3 promoottorin ilmentämä mOX40L ei eronnut kontrollista ja sen todettiin olevan kykenemätön tuottamaan mOX40L hiirisolulinjoissa. Tuloksemme osoittavat, että ihmisen CMV promoottori kykenee ilmentämään transgeenejä hiiren 4T1 ja CT26 solulinjoissa. On kuitenkin huomattava, että isäntälajille natiivien virusten ja promoottorien käyttö olisi tarkoituksenmukaisempaa tulosten käännettävyyden kannalta hiiristä ihmisiin.

Vascular endothelial growth factor C (VEGF-C) is the most studied of the growth factors that control the growth of lymphatic vessels (lymphangiogenesis) and belongs to the same VEGF family as VEGF-A, which controls the growth of blood vessels. The growth of blood vessels and lymphatic vessels is centrally related to the pathophysiology of several cancers that form solid tumours and wet macular degeneration. Unlike VEGF-A, VEGF-C is not currently (2023) a target molecule of any approved drugs, but in clinical trials in the indications mentioned above, combining a VEGF C inhibitor with VEGF-A inhibitors has provided better results than VEGF-A inhibitor monotherapy. The study's objective was converting a phage display library containing single-chain antibody variable fragments (scFvs) screened against VEGF-C into full IgG class antibodies. The scFvs had shown a binding affinity towards the human, mouse, or both VEGF-C variants. The DNA sequences of the best binders of the library had previously been cloned into pLK06H plasmids. The scFvs comprise the variable region of the light and heavy chain (VL and VH) but do not contain the constant regions of the antibody (CL and CHx). Using single-chain antibody fragments as drugs is limited because, in most indications, better stability of whole antibodies, lower immunogenicity, and a longer half-life enabling less frequent dosing is desirable. In addition, the Fc part of whole antibodies often mediates the drug effect, such as complement activation, and whole antibodies are also used as research tools. Secondly, the study aimed to investigate how changing the antibody format affects the binding affinity. To produce whole antibodies, original DNA sequences of pVitro-trastuzumab-IgGk1 plasmid encoding VH and VL regions were replaced with new VH and VL sequences from the phage display library. Several recombinant DNA technology methods were utilised, but the most crucial method was the commercially available NEBuilder HiFi DNA Assembly, which enabled the seamless joining of several DNA fragments into a recombinant DNA molecule in a single-tube reaction. The cloning workflow proved uncertain, as only one constructed antibody production plasmid was sufficiently amplified and expressed in bacterial and mammalian cell cultures. Suboptimal overlapping of DNA fragments and insufficient competence of the bacterial strain used in the transformation were probable bottlenecks. Therefore, as such, the method is not suitable for use on a large scale to convert single-chain antibody fragments into whole antibodies. Also, binding tests were not performed. However, the work done and the antibody production plasmid built is a good basis for further optimisation of the method. In the optimisation, attention should be paid, especially to the quality of the DNA primers and the competence of the bacterial cell line. Also, alternative cloning methods, such as restriction enzymes and ligases, could be used instead of the NEBuilder HiFi DNA Assembly.

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.