Browsing by master's degree program "none"

Background: The World Health Organization (WHO) outlined in their report published in 2014 that antimicrobial resistance (AMR) is a real public health threat worldwide and the actions against it should be taken. Otherwise, the post-antibiotic era where common community-acquired infections can lead to death, could hypothetically become true. The discovery and development of novel antibiotics (ATBs) against Gram-negative bacteria (GNB)-related infections is difficult due to a dual defence mechanism: the extra protection barrier called the outer membrane and efflux pumps which GNB utilize to protect themselves against external noxious compounds. Efflux pumps are expressed at the basal level in GNB, such as E. coli, but when exposed to sub-inhibitory concentrations of ATBs and the intrinsic extruding capacity is exceeded, GNB start overexpressing these “so-called” multi-drug resistance (MDR) efflux pumps. The most abundant and studied MDR efflux pump in E. coli is a tripartite protein complex AcrAB-TolC which traverse through the bacterial cell envelope and is capable of extruding a broad range of structurally unrelated compounds, thus leading to cross-resistance against several classes of ATBs. It has been suggested that antibacterial activity of existing ATBs could be restored again by inhibiting increased efflux activity through efflux pump inhibitors (EPIs). Objectives: Define the optimal assay conditions and a positive control (EPI) to be used in high throughput screening (HTS) of novel EPIs. The assay consists of one E. coli strain of clinical relevance with high intrinsic efflux activity, one ATB and one EPI, both of them at specific concentrations defined during this study. Methods: The intrinsic efflux activities of seven E. coli strains were studied by Hoechst 33342 (H33342) accumulation assay, both in the absence and presence of five commercially available EPIs. The same assay was used in the dose-response studies in which an optimal concentration of EPIs was identified for further to be utilized in the checkerboard assays. The minimum inhibitory concentrations (MICs) were determined by broth microdilution method according to Clinical and Laboratory Standards Institute. The synergistic effects of ATB and EPI in terms of decreasing the intrinsic MIC value of the ATB were determined in the checkerboard assays partially performed by the Biomek i7 Automated Workstation. The data was analysed by using Microsoft Excel and IBM SPSS Statistics, version 25. Results and discussion: E. coli ATCC 25922 had statistically significantly the highest efflux activity of all wild-type pathogenic and non-pathogenic E. coli strains. However, when H33342 accumulation assay was carried out in conjunction with EPIs, E. coli BAA1161 (uropathogenic strain) had the highest median increase in the intracellular level of H33342. Mefloquine showed to be the most potent of all EPIs at the tested concentrations. However, mefloquine increased the intracellular H33342 accumulation even in efflux-deficient E. coli JW5503 (ΔtolC), thus possible additional modes of action or inhibitory activity towards other efflux pumps might exist. Dose-response studies carried out in ΔtolC E. coli JW5503 suggested that CCCP at 1.25 g/ml and mefloquine at 0.5 g/ml were the optimal concentrations. However, for mefloquine, when tested at 0.5 g/ml, the intracellular level of H33342 was not increased in six remaining E. coli strains. Therefore higher concentrations up to ½ MIC were tested in the checkerboard assays. In the antibacterial susceptibility testing, E. coli BAA1161 was the only strain showing resistance to tetracycline and piperacillin, resulting in MIC ratios (MIC wild-type/MIC mutant) of 512 to 2048. Piperacillin and ofloxacin, which showed a MIC ratio of 4 in two E. coli strains, were chosen to the checkerboard assays in which mefloquine reduced the intrinsic MIC of piperacillin by 16-fold and CCCP by 32-fold in E. coli BAA1161. Conclusions: E. coli BAA1161 was chosen to be used as a model strain in HTS due to the highest median increase in intracellular H33342 accumulation and also for being the only strain with resistance towards the ATBs tested. Mefloquine (16 g/ml) was the EPI of choice for the positive control in HTS because the synergistic effects observed between piperacillin and mefloquine were most probably explained by efflux pump inhibition and not by antibacterial activity of mefloquine itself. Piperacillin (256 g/ml) was selected to be used as an ATB in HTS because it was the only ATB which was potentiated by the tested EPIs.

Drug shortages have become a global issue and reasons for drug shortages are several and multifactorial. Definition of drug shortages is not unambiguous. However, in literature are numerous different suggestions to determine the phenomenon of drug shortages. This study provides more focused information on drug shortages and the reasons behind them. The study was performed in cooperation with Orion Corporation. The aim of this study was to explore the in-depth reasons behind medicine shortages from the perspective of one European pharmaceutical company with special focus on Finland, Germany, the United Kingdom and Sweden. Interviews of the company employees were used to achieve this aim and build a few case studies. Further the aim was to investigate in-depth reasons for drug shortages using data from case studies. Case studies were provided by Orion since this enabled use of unpublished information compare the case studies with relevant legal and regulatory measures in the European pharmaceutical framework which influence drug shortages. Reviewing available data from literature and from EUDRA GMDP database for drug shortages and investigate if the data is detailed enough to understand in-depth reasons for drug shortages. Based on the interview results the most common reasons behind drug shortages in Europe are mainly pharmaceutical market structure 38%. It contains many different factors, such as small stock size, local and foreign manufacturing issues, logistics and distribution issues, changes in demand and regulatory issues. However, the manufacturing (33%) or regulatory (29%) reasons are almost as numerous as pharmaceutical market structure issues. Pharmaceutical market structure issues include most common reasons which are categorized in supply-related and demand-related reasons. According to this study supply-related reasons are more common (73%) than demand-related reasons (27%). Some reasons behind drug shortages overlap and often cause a domino effect, whilst other are unique or stand alone, like reasons resulting from natural disasters. The results of this study seem generalizable because the EUDRA GDMP database shows same results and case studies illustrative same reasons behind drug shortages. This study provides more focused information on drug shortages and the reasons behind them from the perspective of pharmaceutical company and authorities.

Cancer afflicts an ever-growing number of people globally each year. In part due to a complex pathophysiology where much is still unknown, the need for new cancer treatments has been persistent, fuelled further by the issue of treatment resistance. An emerging field holding much promise in oncology is immunotherapy, a subgroup of which is oncolytic virus treatments. These treatments utilize the inherent or acquired ability of certain viruses to selectively replicate in tumor cells to fight cancer. One of these viruses is the adenovirus. With these viruses it is possible to modulate the immune response e.g. through the expression of certain genes. The thesis focuses on genetically arming an oncolytic adenovirus in an effort to enhance treatment efficacy. The transgene of choice is the CD40 ligand (CD40L), a costimulatory molecule capable of aiding in the development of systemic antitumor immunity. Adenoviruses have previously been designed expressing the CD40L, however, a novel aspect was introduced with the design and incorporation of a soluble a form of the protein. The main aim of the study was the construction of four functional oncolytic adenoviruses, encoded with either the human or mouse variants of the two CD40L proteins (full-length and soluble). Successful completion required protocols for the cloning, bacterial colony screening, and primary virus production to be established. Insertion of the CD40L transgenes into the E3-gp19k region of the chosen Ad5Δ24 backbone was first attempted with the traditional approach of homologous recombination. The method that ultimately proved successful was a one-step Gibson Assembly® reaction. Screening the bacterial colonies with colony polymerase chain reaction, the potential CD40L positive clones underwent restriction analysis to affirm the presence of the transgene in the viral genome, as well as the retainment of critical elements. Two out of three recombined plasmids carrying the full-length CD40L proceeded to transfection and virus propagation in A549 cells, after which the presence of the adenovirus and CD40L expression was confirmed with immunostaining. Finally, a protocol was successfully established by the construction of one of the intended four viruses. The protocol entails all the main steps from cloning until primary virus production, additionally offering the option of applying it to the genetic arming of the Ad5Δ24 with other transgenes of interest. In terms of future perspectives for the project, following construction of the remaining viruses, the intentions are to validate transgene expression and functionality for all constructs, as well as compare the immunogenicity between the full-length and soluble CD40L. In the event of promising results, the project will hopefully proceed to in vivo studies.

Medication-related events are a significant cause of in-hospital adverse events. Intravenous medication errors occur more frequently and are more likely to result in serious harm than other medication therapies. Closed loop medication management which seamlessly integrates automated and intelligent systems barriers, is used for reducing medication errors. The aim of this systematic review was to identify what kind of scientific studies exist regarding closed loop medication in intravenous medication therapy and barriers related to it. This study is part of a larger systematic review. The literature search indentified 2292 scientific papers. Of these, only 57 were included in the larger review since most of the references were excluded based on titles, abstracts or full-texts. Of these, 21 studies regarding closed loop medication management in intravenous medication therapy were included in this thesis. The studies conserned intelligent infusion devices, computerized physician order entries, clinical decision support systems, automated workflow management systems reducing compounding errors and bar-code confirmation of drugs and patients. According to this review, closed loop medication management potentially reduces medication errors related to intravenous medication therapy. It seems to be more effective to seamlessly integrate the closed loop medication management barriers to each other and to the medication management process than to implement the barriers separately. It’s important to plan the implementation carefully by a multidisciplinary team. In addition, the latest care guidelines need to be taken in to account. Significant resources must be allocated to training and engaging employees and to systematically maintaining and developing the process to manage the successful implementation of the process. This review provides valuable information for Finnish hospitals implementing the closed loop medication management since the concept is not yet well-known in Finland.

Obesity is a health problem linked to Western lifestyle and it is becoming more general. The complexity of regulation of eating makes it difficult to regulate body weight, when multiple neural networks and regions of brain have overlapping functions regarding to energy gain. Sufficient amount of energy is vital for individuals surviving in their living environment. Cholinergic messaging in brain is wide and for example nicotine is known for its appetite reducing effect. Anorexigenic proopiomelanocortin neurons mediate the effect of nicotine. In nucleus accumbens and central nucleus of amygdala extracellular levels of acetylcholine rise during meal, which promotes satiety. Satiety inhibits eating behavior between meals. Amygdala is a part of limbic system and in earlier knowledge it was associated only to regulation of memory, conditioning and fear. Nowadays importance of amygdala in eating behavior research is rising, but most of the studies focus on the effect of cue in regulation of eating. Cholinergic messaging is vigorous in the amygdala and is received from opposite areas of brain between basolateral and central amygdala and therefore this master’s thesis examined the effect of cholinergic messaging in amygdala on regulation of eating behavior. C57BL/6JRcc male mice were stereotaxically implanted with guide cannulas either in the basolateral complex of amygdala (n=10) or central nucleus (n=13). After recovering and habituation to automated pellet dispenser mice were treated with nicotinic and muscarinic receptor agonists and antagonists and eating behavior was recorded for six hours. Nicotine, administered to central and basolateral part of amygdala, lowered the number of pellets mice ate. In central nucleus effect was dose dependent. Mecamylamine had time related effect on eating behavior in basolateral amygdala, but dose dependent response was seen only in cumulative results. Oxotremorine was the only compound which created statistically significant interaction between time and dose. Result was seen in both groups. Scopolamine reduced eating behavior in central nucleus and dose dependency was seen. In basolateral complex scopolamine had time related effect, similar to mecamylamine. The results suggest that amygdala regulates eating behavior even without cue.

Marketing authorized pharmaceutical preparations that are aimed at adult use cause problems both in administration and when dosing. Over and underdosing are the most common medication errors in pediatric population. Only a fraction of medicinal products are clinically tested and evaluated for pediatric use. Children should have the right for the best achievable health, medical care and rehabilitation. The aim of this study was to determine problematic pharmaceutical preparations, formulations or excipients experienced by healthcare professionals. The another aim of this study was to comprise (?) the view of healthcare professionals about 3D-printed medicinal products by using the collected data. By using the data, the problems, challenges, targets for development and other suggestions regarding pediatric medication were identified. New 3D printed medicines suitable for children can be developed by using the observations of this study. The study was carried out as semi-structured interview. Frameworks of the themes were structured by using the subjects of a recently made semi-structured questionnaire. The semi-structured interview was carried out as a group-interview, where the participants were presented open questions according to the themes structured before. According to the study results, the prejudices of the interviewees towards the new technology were mainly positive. The adjustability of the printed medicine by the means of the patient was most highlighted property in the interviews. Accoring to the experiences of the interviewees’, the most suitable pharmaceutical preparations used are liquid preparations such as oral liquids or suspensions. When using solid oral formulations, the age of the patient was not seen as significant. The most common reason for compounding the preparation was the wrong size of the product or dose. The varying availability of pharmaceutical preparations was seen as delaying factor at the start of the medical treatment. In the interview the pharmacists recognized the most common excipients causing adverse events. The different roles of the occupational groups were identified according to their work duties.

Currently, there is an undeniable need for more effective treatments of depression. The efficacy of traditional antidepressant drugs becomes apparent after multiple weeks of treatment. New advancements in depression treatments have been made, as glutamatergic NMDA-receptor antagonist ketamine is seen to ameliorate symptoms rapidly, even only hours after drug administration. Understanding ketamine’s mechanism of action as an antidepressant could enable the development of more effective antidepressant drugs. The critical molecular level component in ketamine’s antidepressant effect is considered to be the activation of TrkB tyrosine receptor kinase B, which subsequently leads to the initiation of signaling pathways, which regulate synaptic plasticity. So far, it has not been examined; whether there is a difference in ketamine’s antidepressant effect based on the dosing-time of day. The aim of the present study was to find out if there is a variation between ketamine’s effect on synaptic plasticity and the circadian phase in which the drug is administered. Ketamine’s (200 or 50 mg/kg, i.p.) effects were studied in C57BL/6J–mice during light phase (mouse’s inactive phase) and dark phase (mouse’s active phase) of the day. The phase of the day didn’t affect the activity of TrkB signaling in its related parts (pTrkBTyr816, pGSK3βSer9, p-p70S6KTyr421/Ser424 and p-p44/42MAPKThr202/Tyr204) in prefrontal cortex samples which were analysed in Western blot assay. Ketamine increased dose-dependently the phosphorylation of GSK3βSer9 and p70S6KTyr421/Ser424 as well as decreased p-p44/42MAPKThr202/Tyr204 at 30 minutes after drug administration in both phases of the day. Ketamine (200 mg/kg, i.p.) also lowered the glucose concentration measured from the trunk blood. To examine the effect of hypoglycemia on the activity of TrkB signaling another experiment was conducted. The hypoglycemia induced by insulin detemir (6 IU/kg, i.p.) didn’t affect any measured protein phosphorylation at 60 minutes after drug administration. The results of this study support the notion of ketamine’s rapid and dosedependent induction of neuroplasticity. The possible role of hypoglycemia in ketamine's neuropharmacology should be investigated in future studies.

Inappropriate polypharmacy refers to a situation where more than appropriate amount of medicines are used by a patient. Aged people with multiple morbidities and medications use a lot of health care services and are thus especially vulnerable to iatrogenesis, the health hazards resulting from the acts of a health care system. As a part of normal ageing, geriatric syndromes (e.g. falls, delirium and urinary incontinence) are clinical conditions and symptoms crossing several organ systems and they cannot be connected to a certain individual disease. Geriatric syndromes complicate recognition of adverse drug reactions on aged. This increases the risk of prescribing cascade, where medicines are prescribed to treat adverse drug reactions caused by another medicine. In this master´s thesis the root causes for inappropriate polypharmacy and drug-related problems (DRP) with home-dwelling aged were researched retrospectively from the viewpoint of risk management. Research method was based on root cause analysis (RCA) that was simplified suitable for this research. Research material was based on an intervention research conducted in 2015– 2017 on home-dwelling aged receiving regular home care from the City of Lohja, Finland. In the intervention research, a coordinated community-based medication management model for home-dwelling aged in primary care was developed to identify homedwelling aged with clinically significant drug-related problems. As research material, there were five (n=5) patient cases used who received comprehensive medication review (CMR) in the intervention research to solve their drug-related problems. The research material composed of individual patient interviews conducted at patients’ homes as a part of their CMR visits. Also, the nurses (n=3) of home care and physicians (n=2) from local health centres having participated in the treatment of the home-dwelling aged in question, were interviewed individually. Markings made in the patient records were utilized as well as research material. The interviews of the nurses and physicians were recorded, transcribed and analysed with inductive content analysis considering principles of root cause analysis. According to the nurses and physicians, central clinically significant medication-related problems with home-dwelling aged are various prescribing care parties, multiple medications, the increased use of over-the-counter (OTC) medicines and natural products, the uncertainty of health care professionals of the medication of a home-dwelling aged as well as the occurrence and medication of pain and sleeping disorders with aged. Other essential problems related to the health care system are various attending physicians, obscurely recorded medication data in patient record system, the use of benzodiazepines and other psychopharmaceuticals and ignored renal function in medicine dose adjustment. Problems related to home-dwelling aged are attachment for medicines, resistance to change and desire to take care of their own medication. In addition, memory disorders and vertigo were mentioned as problems related to the medication of aged. Seven root causes for inappropriate polypharmacy and drug-related problems were observed: lack of health care resources, segmented treatment between various health care parties, varying skills and knowledge of health care professionals, ambiguous division of responsibilities between health care professionals, challenges in communication between different care parties, the heterogeneity of patient record systems and problems related to their use as well as the knowledge, opinions and personal situation of a home-dwelling aged. Based on the research, the medication of home-dwelling aged should be improved by striving for centralizing care in one physician either on private or public health care. Among home care nursing personnel there is a need for additional training on medications and pharmacists should participate in regular medication reviews for home-dwelling aged. Patient record systems and data transmission between them should be improved and medication data should be recorded more precisely. Cooperation and communication between home care and health centre should be developed and the division of responsibilities should be clarified. Participation of the home-dwelling aged and their relatives in the care should be promoted. Furthermore, geriatric expertise should be utilized better in the care of the home-dwelling aged.

Breast cancer is the most common cancer in women worldwide and the number of new events is on the increase. Like many other serious diseases, breast cancer reduces patient’s health related quality of life (HRQoL) and breast cancer treatment burdens our society. Examination of breast cancer patient’s HRQoL makes it possible to calculate how effective breast cancer treatments are. Nevertheless, only cost-effectiveness analysis would further help us allocate the resources of our society in the best way possible. The aim of this study was to produce research about breast cancer treatment’s effects on patient’s HRQoL and to compare generic 15D- and EQ-5D-5L-instruments. The results can be used in the future research and the study might be useful, when it’s time to develop international protocol for measuring HRQoL. The study population included 152 breast cancer patients who were treated in HUCH and whose HRQoL were measured by 15D-, EQ-5D-5L- and VAS-instruments. All measurements were done twice, first before the treatments and then six months after the beginning of the treatments. 89 (58.6 %) patients answered both 15D-questionnaires and 81 (53.3 %) patients answered to both EQ-5D-questionnaires. 57 (37.5 %) patients didn’t respond to any questionnaire. Only some background information was available of this population. The average HRQoL for breast cancer patients’ was 0.92 before the treatments and 0.88 six months after the beginning of the treatments when measured by 15D. The same average HRQoL was 0.86 before the treatments and 0.80 six months after the beginning of the treatments when measured by EQ-5D-5L. During six months’ period, patients HRQoL reduced (-0.04) when it was measured by 15D and (-0.06) when it was measured by EQ-5D. The changes of HRQoL were clinically important (The minimum important change, MIC > ± 0,015) when measured by 15D. HRQoL reduced more with patients who received a mastectomy than with patients, who received a breast conserving surgery according to both instruments. According to the results, the chosen instrument has an effect of breast cancer patients’ HRQoL. It means that the chosen instrument also has an effect of treatment’s effectiveness. 15D offers higher HRQoL values, but EQ-5D offers a greater change in patient’s HRQoL. HRQoL was measured by two different generic instruments in two different times, which was assumed to be the strength of this study. The new 5L-version of EQ-5D-instrument was also used. This is possibly the first time, when 5L is used in this type of study.

Formulation development for protein drugs should base on the knowledge of the mechanism of protein degradation. Excipients and formulation can be chosen to stabilize the protein and prevent decomposition. Stability testing is important to identify the likely degradation routes and provide information for formulation development and stability-indicating analytical method development. Gonadotropin-releasing hormone (GnRH) is a neuropeptide hormone that regulates the synthesis and release of gonadotropins: luteinizing hormone (LH) and follicle-stimulating hormone (FHS). Analogs of the endogenous GnRH have been developed to achieve more potent and longer-acting agonists or antagonists. GnRH agonists degrade in several pathways. The primary degradation routes are hydrolysis/backbone cleavage, oxidation, isomerization and aggregation. The stability of GnRH agonists in solid dosage forms has not been studied as excessively as in solutions. The objective of this study was to evaluate the stability of a GnRH agonist (API) at different storage conditions in powder form and in tablet formulations with maize starch or hydroxypropyl methylcellulose (HPMC). The samples were stored for three months at 5 °C (common refrigerator conditions) 25 °C/58 %RH (long-term conditions), and 40 °C/75 %RH (accelerated storage conditions). The samples were analyzed using high performance liquid chromatography. Additionally, the mechanical properties of the formulations and tablets were studied. The stability of API was confirmed in tablet dosage form, when maize starch or HPMC were used as excipients. No degradation products of API were found. As a pure powder API did not degrade either, but it did not stay physically stable at 40 °C/75 %RH. Stressed conditions could be used to find out degradation products in solid state that were not found in this study. Further, the formulations were not ideal, because neither of the studied excipient produced tablets with desirable properties.