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The obligatory storing of medicines is a vital part of the secure supply of medicines in Finland. Over the past few years, its importance has further increased due to the growing number of medicine shortages. Evaluating the effectiveness of the obligatory storing system is important in order to improve it, but so far research on the matter has been limited. The aim of this study was to investigate how the obligatory storages of pharmaceutical manufacturers and importers are used during medicine shortages in Finland, and to assess the effectiveness of their use in these situations. The material for this study consisted of medicine shortage notifications which had been received by the Finnish, Swedish and Norwegian medicine authorities with a forecasted starting date between January and June 2022. In addition, Finnish exemption permits for lower storage levels from the same time period were investigated. Medicine shortage notifications were grouped based on the obligatory storing status of the medicine. The share of obligatorily stored medicines out of all shortage notifications was the smallest in Finland (10%) when compared to Sweden and Norway. There were no statistically significant differences in any of the countries in the duration of a shortage between obligatorily stored medicines and medicines which are not obligatorily stored. In total, 151 exemption permits had been granted within the time period of the study, 129 (85%) of which did not have a coinciding medicine shortage. This suggests that a patient-affecting shortage had successfully been avoided. The remaining 22 exemption permits were linked to a shortage which started either prior to, or during the validity of the exemption permit. In the Finnish data, 91 notifications concerned obligatorily stored medicines but in 69 (76%) of these cases no exemption permit had been applied for or been granted in relation to the shortage. The results of this study indicate that the obligatory storing of medicines was used to respond to several medicine shortages during the first half of 2022, and in most cases, it seems to have been an effective way to avoid a patient-affecting shortage. However, in some cases the use of an exemption permit was not well-timed, a shortage was experienced despite the releasing of products from the storage, or obligatory storages were not used at all. Based on the results, further research on the practices of obligatory storing and the factors which affect the use of exemption permits is needed to develop the system further and to improve its effectiveness in responding to medicine shortages.

During the past few decades, the explosion of discovery in cancer and immunological research has led to the increased understanding of the interactions between the immune system and tumors. These developments have provided vital information about the immune system’s role in cancer development. It is evidenced that the immunity system is capable to distinguish tumor cells from normal tissue by recognizing tumor antigens that are exclusively expressed on tumor cells or are presented in greater amounts on tumor cells than normal cells. Consequently, the immune cells start to attack tumors for protecting the host. The possibility to use the immune system as a weapon against cancer cells leaded to the promising innovation – cancer immunotherapy – which aims to activate the body’s own immune system and its components to mount antitumor immune responses for eliminating cancer cells. The antitumor efficacy and high safety profile of several immunotherapeutic strategies have already been demonstrated thereby resulting in their integration into clinical practice. However, most patients have not benefited from cancer immunotherapy as a single treatment. In this regard, new innovative methods are clearly needed to overcome the obstacles hindering the clinical success of this field. Therapeutic cancer vaccines are emerging as attractive immunotherapies currently being evaluated in both pre-clinical and clinical studies. The purpose of cancer vaccines is to eradicate tumor cells by eliciting antitumor CD8+ T cell responses against the injected tumor antigens. Due to the ability to specifically kill tumor cells and simultaneously trigger immune responses against tumor antigens via direct oncolysis and by encoding transferred tumor antigens, oncolytic viruses are of significant interest for being used as in situ cancer vaccines. Despite these unique properties, several factors such as tumor immunosuppression and immune tolerance to targeted tumor antigens resembling antigens of normal tissues hamper the use of oncolytic vaccines in clinic. Instead of focusing only on CD8+ T cells, it has been suggested that giving more attention to CD4+ T helper cells, which are required for priming and expansion of CD8+ T cell responses, could be the key to improve the efficacy of cancer vaccines. Researchers have also demonstrated that an ongoing antigen-specific CD4+ T cell response can lead to the bystander activation of surrounding T cells with unrelated antigen specificities. Based on this theory, the hypothesis of this study was to employ the pre-existing immunological CD4+ memory against infectious pathogens in generating bystander CD8+ immunity against solid tumors. In this study, mice transplanted with poorly immunogenic B16-OVA tumors were pre-immunized with the chosen vaccine to induce immunological CD4+ memory against an infectious pathogen. Tumors were then treated with already developed cancer vaccine, which was peptide-coated conditionally replicating adenovirus (PeptiCRAd) complex. PeptiCRAd was constructed by electrostatically coating adenovirus with both pathogen-derived and tumor-derived peptide. The intratumorally injected double-coated PeptiCRAd complex was assumed to activate peptide-specific T cells and thus, result in anti-pathogen CD4+ T cell recall responses and the following bystander activation of antitumor CD8+ T cells, which can then mount an effective immune response to destroy cancer cells. The efficacy of this treatment was observed in pre-immunized mice by measuring the growth of injected tumors. The experiment was repeated identically with non-immunized naïve mice to see the difference in the results. The immunological background of this treatment approach was investigated by analyzing mouse tissue samples with standard immunological techniques including ELISA, IFN-γ ELISPOT and flow cytometry. This study showed that long-term immunological memory against the pathogen was successfully accomplished and the strongest inhibition of tumor growth in pre-immunized mice was achieved with double-coated PeptiCRAd, whereas the antitumor efficacy was not seen in naïve mice. Additionally, a new ex vivo method to detect pathogen-specific CD4+ T cells from spleen was developed and the stimulation of cell-mediated immunity by this treatment was supported by finding the highest levels of pathogen-specific CD4+ Th1 cells from mice treated with double-coated PeptiCRAd. Some encouraging results concerning the beneficial immune cell composition of tumors and tumor draining lymph nodes were also obtained from other performed experiments. Though further immunological analyses are required for understanding the precise mechanisms of action behind the treatment, the increased immunogenicity and antitumor efficacy of double-coated PeptiCRAd can still be considered as a consequence of the bystander effect, which can possibly be utilized for developing improved strategies to win the fight against cancer.

There is a growing interest towards continuous manufacturing in pharmaceutical industry. When considering solid dosage form manufacturing and continuous wet granulation, twin screw granulation is the most studied technology. In spite of this, process knowledge is not as comprehensive as it is for batch granulation technologies such as high shear granulation. One problem with twin screw granulation seems to be bimodal and broad particle size distribution of granules. Specific causes of bimodal distribution and the ways to possibly gain unimodality either in granulation step or via dry milling are not fully understood. The purpose of this study was to optimize the ConsiGma25 continuous twin screw wet granulation process and study dry milling as a possible way of gaining unimodal particle size distribution in order to optimize particle size for tablet compression purposes. One aim was also to compress tablets from the obtained twin screw granules and evaluate the quality against high shear and direct compressed tablets of same formulation. Central composite circumscribed design was used as a design of experiments for twin screw wet granulation process. Factors (powder feed rate, screw speed, liquid to solid ratio (L/S ratio) and mill screen size) were varied in two levels. In total, 29 experiments were conducted. Tablet compression design was fractional as only 11 experiments from the twin screw granulation design were tableted. High shear granulation was conducted with Diosna P-1/6 using three different L/S ratios. All of these batches were compressed to tablets. Also direct compression was carried out. Formulation used had ibuprofen as active pharmaceutical ingredient, mannitol and MCC as fillers, HPC as binder and croscarmellose sodium as disintegrant. Lubricant, sodium stearyl fumarate, was blended to granules before tablet compression. Torque was measured during granulation in order to evaluate equilibration of the twin screw granulation process. It stabilized quickly and stayed stable after parameter changes and increased as L/S ratio and barrel fill rate increased. Particle size distribution and flowability of granules were analysed and tablets were analysed according to European and United States pharmacopoeias. Also tensile strengths and compactibilities were evaluated. Particle size distributions of unmilled twin screw granules were bimodal and broad. After dry milling, QicPic dynamic image analysis results showed unimodal particle size distributions for all experiments whereas Mastersizer laser diffraction analysis showed more unimodal distributions for experiments milled with smaller mill screen sizes. Mill screen size had the largest effect on particle size and as increased mill screen size increased particle size. Results showed that dry milling was a way to optimize particle size distribution for tablet compression purposes. Also flowability of formulation and process parameters needed to be optimized as granulation parameters had an effect on particle size and manufacturability was enhanced with better flowing formulation compared to previous study. Knowledge of the influence of L/S ratio, screw speed and powder feed rate, on granule size was gained. The effect of these process parameters varied depending whether d10, d50 or d90 was measured and particle size analysis method used. Increased L/S ratio and screw speed increased granule size as increased powder feed rate decreased it. Twin screw tablets showed higher tensile strengths and better compactibility compared to both high shear tablets and direct compressed tablets. Twin screw tablets showed also faster dissolution compared to direct compressed tablets probably due to of lower compression force. Mill screen size had the largest effect on dissolution properties of the twin screw tablets. When larger mill screen size was used, dissolution was slower due to larger particle size. Also increased powder feed rate made dissolution rate slower due to higher fill rate of the barrel. In other tablet properties analysed, no significant differences were seen between different twin screw experiments or between twin screw tablets and direct compressed tablets. All of the twin screw tablets and direct compressed tablets fulfilled the requirements of European and United states pharmacopoeia. As a conclusion, continuous granulation process was successfully optimized and high quality tablets resulted showing especially the effect of dry milling on granule size and shape as well as on tablet properties.

Orexin receptors have gained more attention due to increased knowledge of their physiological significance. The successful development of orexin receptor antagonists treating insomnia has enlarged the scope of research leading to an interest in developing small molecule agonists that have drug-like properties and induce activation in the orexinergic system. Transforming this idea into reality has remained an unaccomplished challenge. In this work, molecular mechanism of activation in orexin receptor type 2 is studied by conducting molecular dynamics simulations after capturing coordinates of active and inactive state crystal structures. The crystal structure coordinates define the starting pose for the protein and the ligand in the simulations. Preliminary steps prior to simulation include building the surrounding system and model building in which homology modeling is employed to reconstruct missing loops. A 100-nanosecond simulation is executed for both models. Active and inactive state models display stable binding event characteristics when examining the coordinate changes of every atom during the simulation. No major conformational changes are observed. The most congruent feature relative to the literature is the difference in the interactive role of residue Q3x32 between the simulations. The agonist forms two consistent hydrogen bonds with the upward-shifted Q3x32 while an inactive downward-facing version is observed in the antagonist model. Some residues present unexpected features omitting comparative functions of literature, which along with general inconsistency of protein-ligand contacts undermine the reliability of models heavily. The simulations conducted need to be extended to produce a hypothesis for the activation mechanism because of the defects around simulation protocols and the low quantity of simulation runs.

Children´s medication treatment has many special features that predispose to medication errors, such as dosing of medications according to weight or age and the off-label use of medications. In the medical treatment of children high-alert medicinal substances are used and the incorrect use of which can cause harm to the patient. The aim of this study was to identify medication errors in pediatric patients of parenteral nutrition products (PN) and concentrated electrolytes, which belong to high-alert medicinal substances in different stages of medication management and use process and also to identify the contributing factors behind the errors in order to promote medication safety. The data for the retrospective registry study were made up of HaiPro accident reports (n=528) related to PN, lipids, concentrated electrolytes, solutions affecting electrolyte balance and dialysis fluids made in the period 2018-2020 at the Children`s and Adolescent`s hospital in Helsinki from which the reports related to high-alert medications were identified (n=317). ISMP´s (Institute for Safe Medication Practices) and JCI´s (Joint Comission International) lists of high-alert medications was used to limit the data. The final research material was further limited to reports (n=254) in which the medicinal substance appeared more than ten times in the entire material. The data were analyzed quantitatively to describe the frequencies (n) and percentages (%) of PN and concentrated electrolytes, and qualitativevely to identify the stages of the medication management and use process, types of medication errors and contributing factors. High-alert medications accounted for more than half (n=317/528, 60,0 %) of the entire material of this study. Medication errors (n=378) were identified most during the administration and preparation phase of the medication. In the administration phase, 56,8 % (n=117/206) of errors were identified with PN and the most common error was disturbances in the infusion tubing, wrong infusion rate or wrong dose. With concentrated electrolytes, errors in the administration phase were identified in 50,0 % (n=86/172) of all errors and the most common error was wrong product the patient received, wrong infusion rate and medicine not being administered. In the medication preparation phase, errors were identified in 20,9% (n=43/206) of PN and 30,2% (n=52/172) of concentrated electrolytes. The most common error in the preparation phase was incorrect preparation of medicine with both groups of medicinal substances. Factors related to workload and resources and human factors related to the employee, were most identified as contributing factors (n=753) in both medication groups. Targeting preventive protections, especially in the administration and preparation phases of the medicine is desirable both with PN and concentrated electrolytes. It is also important to plan safeguards comprehensively for the entire mediacation management and use process taking into account the key contributing factors that predispose to medication errors.

Parkinson's disease is a neurodegenerative disorder where dopaminergic neurons in substantia nigra are gradually destroyed. Less than 10% of Parkinson's disease cases are genetic. For example mutations in α-synuclein, LRRK2-, parkin-, PINK1- and DJ-1 are known to cause Parkinson's disease. There is still no curative treatment for Parkinson's disease. Alpha-synuclein is linked to Parkinson's disease through Lewy bodies. Three point mutations causing Parkinson's disease have been found in a gene coding α-synuclein. Alpha-synuclein has been expressed in Drosophila melanogaster, C. elegans and mouse. Main function of LRRK2-protein is thought to be kinase activity. Mutations in LRRK2-gene are the most common known cause of Parkinson's disease. LRRK has been expressed in Drosophila melanogaster, C. elegans and mouse. LRRK2 knock-out Drosophila melanogaster and mouse have also been studied. Parkin is a neuroprotective protein and its deficiency results in a loss of neurons in substantia nigra. Mutations in Parkin cause 50% of recessive Parkinson's disease. Parkin knock-out Drosophila melanogaster and mouse and Drosophila melanogaster and mouse expressing human Parkin are Parkin animal models. PINK1 is a mitochondrial protein coded by nucleus. DJ-1 is thought to have a part in mitochondria maintenance and protection. Both PINK1- and DJ-1 knock-out Drosophila melanogaster and mouse have been studied. None of the genetic animal models of Parkinson's disease is identical to symptoms and pathology of human Parkinson's disease. The purpose of the experimental part of this thesis was to examine non-drug induced behavioural test and Cerebral dopamine neurotrophic factor (CDNF) in 6-OHDA lesioned rats. CDNF protects and restores dopaminergic neurons. The non-drug induced behavioural tests included in this study were stepping test, cylinder test and staircase test. An old and widely used drug induced test for Parkinson's disease, amphetamine-induced rotation test, has problems that have led to a seek for replacing and complementary test methods. In amphetamine-induced rotation test dopamine agonist is given to a unilaterally 6-OHDA lesioned animal. The agonist causes rotational behaviour that can be measured with designed equipment. The stepping test measures forelimb akinesia in rats. In the experimental setting the rat is moved sideways when it is held only one front paw on a table and adjusting steps are counted. In the cylinder test front paw preference is measured. In the experimental setting the rat is placed in a transparent cylinder and the front paw preference is counted on rears and on ground contacts after a rear. The staircase test measures front paw coordination and function. In the experimental setting the number of sucrose pellets picked up from a double staircase is counted. There were no significant differences between lesioned groups in stepping test, cylinder test or in staircase test. It is possible that the 6-OHDA lesion used in the experiment was not extensive enough. Different non-drug induced behavioural tests supplement each other and they should be combined for the best result. Combining different behavioural tests enables more reliable results and versatile information than the amphetamine-induced rotation test alone.

Part I: Parkinson's disease is a slowly progressive neurodegenerative disorder in which particularly the dopaminergic neurons of the substantia nigra pars compacta degenerate and die. Current conventional treatment is based on restraining symptoms but it has no effect on the progression of the disease. Gene therapy research has focused on the possibility of restoring the lost brain function by at least two means: substitution of critical enzymes needed for the synthesis of dopamine and slowing down the progression of the disease by supporting the functions of the remaining nigral dopaminergic neurons by neurotrophic factors. The striatal levels of enzymes such as tyrosine hydroxylase, dopadecarboxylase and GTP-CH1 are decreased as the disease progresses. By replacing one or all of the enzymes, dopamine levels in the striatum may be restored to normal and behavioral impairments caused by the disease may be ameliorated especially in the later stages of the disease. The neurotrophic factors glial cell derived neurotrophic factor (GDNF) and neurturin have shown to protect and restore functions of dopaminergic cell somas and terminals as well as improve behavior in animal lesion models. This therapy may be best suited at the early stages of the disease when there are more dopaminergic neurons for neurotrophic factors to reach. Viral vector-mediated gene transfer provides a tool to deliver proteins with complex structures into specific brain locations and provides long-term protein over-expression. Part II: The aim of our study was to investigate the effects of two orally dosed COMT inhibitors entacapone (10 and 30 mg/kg) and tolcapone (10 and 30 mg/kg) with a subsequent administration of a peripheral dopadecarboxylase inhibitor carbidopa (30 mg/kg) and L- dopa (30 mg/kg) on dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens of freely moving rats using dual-probe in vivo microdialysis. Earlier similarly designed studies have only been conducted in the dorsal striatum. We also confirmed the result of earlier ex vivo studies regarding the effects of intraperitoneally dosed tolcapone (30 mg/kg) and entacapone (30 mg/kg) on striatal and hepatic COMT activity. The results obtained from the dorsal striatum were generally in line with earlier studies, where tolcapone tended to increase dopamine and DOPAC levels and decrease HVA levels. Entacapone tended to keep striatal dopamine and HVA levels elevated longer than in controls and also tended to elevate the levels of DOPAC. Surprisingly in the nucleus accumbens, dopamine levels after either dose of entacapone or tolcapone were not elevated. Accumbal DOPAC levels, especially in the tolcapone 30 mg/kg group, were elevated nearly to the same extent as measured in the dorsal striatum. Entacapone 10 mg/kg elevated accumbal HVA levels more than the dose of 30 mg/kg and the effect was more pronounced in the nucleus accumbens than in the dorsal striatum. This suggests that entacapone 30 mg/kg has minor central effects. Also our ex vivo study results obtained from the dorsal striatum suggest that entacapone 30 mg/kg has minor and transient central effects, even though central HVA levels were not suppressed below those of the control group in either brain area in the microdialysis study. Both entacapone and tolcapone suppressed hepatic COMT activity more than striatal COMT activity. Tolcapone was more effective than entacapone in the dorsal striatum. The differences between dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens may be due to different properties of the two brain areas.

Cancer immunotherapies aim to target the immune defence mechanisms of the body specifically and efficiently against the tumour tissue. Cancer vaccines and oncolytic viruses are forms of active immunotherapies, which require patients having a properly functioning immune system. The vaccines are based on the administration of tumour antigens into the body to which the immune system reacts. However, often the response is not robust enough. The oncolytic viruses in turn kill the cancer cells which causes the release of antigens from the tumour tissue. Viruses usually elicit a strong immune response but sometimes it is targeted too much against the virus instead of the tumour. Oncolytic vaccine is a composition of an oncolytic virus and a cancer vaccine. Tumour antigens can be coded to the genome of the virus therefore, when the virus invades tumour cells they start to produce the antigens. Eventually the cancer cells are also destroyed due to viral replication. The antigens can be tumour-associated that is, they are expressed in healthy tissues too. Their usage is not always efficient which is why an interest towards utilizing tumour-specific antigens has been increased. Considering the expression of antigens, tumour tissue is very heterogenous and distinctive between patients. Hence, utilizing mutated patient unique neoantigens would enable the development of personalized tumour-specific oncolytic vaccines. Genetic modification of viruses is complicated thus, an easier way to insert the neoantigens to the virus has been invented. The developed oncolytic vaccine platform is called PeptiENV, and it is designed to use with enveloped viruses. The idea is to fuse tumour-specific antigens onto the envelope of the virus and eliminate the need of gene insertion. The aim of this study is to investigate in vivo the efficacy of PeptiENV in preventing tumour growth and eliciting a tumour-specific immune response. An object is also to observe survival times of the treated animals. Furthermore, the preservation of infectivity is studied in vitro. The research was executed with two potential oncolytic viruses, vaccinia virus (VACV) and herpes simplex virus type 1 (HSV-1). The PeptiENV complex was formed by using an artificial tumour antigen, ovalbumin epitope SIINFEKL, which was attached to the viral envelope with cell penetrating peptide (CPP) or cholesterol anchor. The preservation of infectivity was examined by measuring cell viability of PeptiENV infected cells. Animal experiments instead were performed with a mouse melanoma model created with B16-OVA cells, which express ovalbumin and therefore the antigen epitope SIINFEKL. PeptiENV was compared to control treatments which were virus, SIINFEKL peptide and complexation medium only. Treatments were administered as intratumoural injections. Tumour growth was followed by measuring the size of implanted tumours every other day. With flow cytometry, tumour-specific immune response was assessed by acquiring the relative amount of SIINFEKL-specific CD8+ T cells in the tumour tissue. Euthanizing dates were registered in order to observe the survival of the mice. According to the in vitro results, conjugation of peptides to the virus does not affect infectivity. In addition, the in vivo studies show that PeptiENV VACV CPP prevents tumour growth the most. Difference in tumour growth between PeptiENV VACV CPP and control treatments is significant. Mice injected with the same treatment also lived considerably longer than mice injected with virus, peptide or medium only. Also, PeptiENV HSV-1 hinders tumour growth distinctly more than virus only and slightly more than SIINFEKL only, but unfortunately it did not have an evident impact on the survival time. In both experiments, the PeptiENV treatment elicits the largest proportional amount of SIINFEKL-specific CD8+ T cells. In other words, PeptiENV engenders a tumour-specific immune response. In the PeptiENV VACV study the difference to control treatments is clearer than in the PeptiENV HSV-1 study. At present, the PeptiENV platforms performs better with VACV than HSV-1. With further investigations however, the results can be verified and improved. All in all, the results are encouraging. The PeptiENV platform shows great promise for being a part of personalized cancer immunotherapy developments in the future.

Medication safety is safety related to the use of medicines. Medication errors are drug treatment related events which can lead to medication safety incidents. Medication process is multi-professional teamwork which contains a risk of medication error on every step. It is important to identify potential safety risks in order to prevent the risk events. Medication errors can occur for example during the transfer of the prescription information in to the medication list. It is important that medication lists are accurate and up to date so that patient's medication therapy is optimal. The aim of this study was to assess whether the primary health care medication lists are up to date, accurate and easy to read. The aim was also to identify what kind of information in medication lists was open to interpretation. In one of the municipalities, the medication lists at home were compared to the medication lists at primary care to see whether both lists had identical information on the medication. The data of this study consisted of 240 medication lists from primary care units in three Finnish municipalities. The lists contained altogether 3062 medications. Most of the lists were printed from the patient information systems. Some of the lists were copied from the home medication lists. All medication lists were systematically reviewed and issues open to interpretation were documented in a structured Microsoft Excel table. The data were transferred to SPSS 20 Program for statistical analysis. Most (73%, n=174) of the medication lists (n=240) were incomplete. One-fifth (n=612) of the medications in use (n=3062) contained missing information on medicines. The total number of discrepancies was 807 (mean 3.4 discrepancies per medication list). The most common discrepancies were related to the time of administration (n=277) and dosages (n=241). Duplicate medications included a lot of confusing information. Discontinued medications were not always clearly marked. In only one of the municipalities the medication lists had a space for marking the indication. There were some differences between home medication lists (n=62) and primary care medication lists. ™ Based on this study medication lists have a lot of discrepancies and ambiguities in their information content. The medication lists do not always accurately tell the patient's current medication. Interpretation of inaccurate medication lists consumes unnecessarily doctors' and nursing staff's time. Inaccurate medication lists are a risk to patient safety. It is also important that the medication lists would be similar in all health care units. Electronic prescriptions, the National Health Archive and medication list developed by Information Management Service of Healthcare are expected to solve at least some of the problems related to medication lists.