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Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.

Solid materials can exist in two major forms: in crystalline or amorphous form. Amorphous form is defined as no long term order existing in solid structure in molecular scale. Amorphous materials have different physicochemical properties compared crystalline forms of same substance. Amorphous materials doesn't have sharp melting point as crystalline materials. When heated above so called glass transition temperature amorphous materials become rubbery (plasticization) and when cooled below they become glassy (hard and brittle). Amorphous forms can also have different dissolution properties which makes them useful in formulation of poorly soluble drugs. Amorphous forms are less stable compared to crystalline form. That's due amount of free energy stored in it's structures. Amorphous materials can be manufactured in many ways including quench cooling, hot-melt-extrusion, spray drying and lyophilisation (freeze drying). In experimental section effect of grinding method in properties of amorphous indomethacin was studied. Amorphous indomethacin was prepared by quenching of melt in liquid nitrogen. Properties of amorphous indomethacin was studied by x-ray powder diffraction and differential scanning calorimetry. Measurements were performed in different time stamps varied form 0 to 92 days. Measured properties were crystalline content, glass transition temperature, change in heath capacity, heat of crystallization, heat of melting and melting points of crystallized forms. Calorimetry data was recorded only from totally amorphous samples. It can be seen in results that different patches are not comparable statistically but when comparing room temperature ground and liquid nitrogen ground samples to each other differences can be found in every set. Difference is observed in initial time of crystallization (time when crystallinity can be measured first time) and in thermodynamical properties such as change in heat capacity, glass transition temperature and heat of melting. Solid dispersions of indomethacin and xylitol were prepared in 3 different compositions (5%, 10% and 20% xylitol in indomethacin). XRPD and DSC data were measured at different time stamps (aged 1 to 63 days). 5% and 10% dispersions found to be stabile and being amorphous in all time stamps. 20% dispersion was already partly crystallized at 63 days (especially liquid nitrogen ground sample).

Today, many of the new drugs are poorly soluble in water, which can be a problem in the drug development. Solid dispersion is a formulation technique, which improves the dissolution rate of the drug. However solid dispersions, where the drug is in amorphous form, are often unstable. Because of that, solid dispersions, where the drug is in crystalline form, have been developed. Drug crystallization and factors affecting to the crystallization, such as amount of the polymer, are important to examine to be able to develop better drug products. Different kinds of mathematical models, which describe the kinetics of crystallization, has been developed to help to understand the crystallization event more comprehensively. In this study, the crystallization of the amorphous drug, in the absence of polymer and with a low polymer concentration, was investigated. The crystallization was also examined using a mathematical model designed to determine the kinetics of crystallization in order to find out does it work in this case. A model drug was felodipine and polymers used in this study were HPMCAS-LF and PVP K30. The concentration of polymers in the solid dispersions was 10% and 20%. It was found that a small amount of polymer has a very significant effect on crystallization rate of felodipine. Mathematically defined crystallization rate constant k increased by 13 times, when the amount of PVP was decreased 20 % to 10 %. The polymer concentration also had an effect on nucleation time which is the time before crystallization occurs. For example in the solid dispersion, where PVP concentration was 10 %, the nucleation time was five times slower and 20 % PVP consentration ten times slower than felodipine alone. The work also showed that HPMCAS stabilizes the amorphous state of felodipine better than PVP at 40 ° C / 75% RH conditions. This was observed in both MTDSC-measurements and the polarizing light microscopy. The difference between polymers was thought to be due to weakening of the interactions between PVP and felodipine by the influence of water in humid conditions. However, the different formulations had no significant effect on dissolution characteristics of felodipine. There is a possibility that felodipine crystallizes at the beginning of dissolution. It should be noted that mathematical method tested was not able to model crystallization kinetics properly in this study. So care should be given, when using a mathematical model in the product development.

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.

Diseases of the posterior eye segment, such as age-related macular degeneration (AMD), diabetic retinopathy, diabetic macular edema and glaucoma are the leading cause of blindness worldwide. Current therapy to treat these vision-threatening diseases relies on intravitreal injections to maintain a desired therapeutic drug concentration in the back of the eye. Frequent intravitreal injections are uncomfortable with poor patient compliance and causes major burden to the healthcare systems as well as to the patients. Small molecule drugs have shorter half-life in the vitreous and are eliminated rapidly. This requires frequent intravitreal dosing intervals that are not feasible in the clinical settings. Also, intravitreally injected small molecule drugs are often poorly and non-specifically distributed to the ocular tissues causing adverse effects. To address these issues, controlled and sustained drug delivery systems in the form of drug conjugates are desirable. Conjugating small molecule drugs with enzymatically cleavable peptide linkers increases the residence time in the vitreous. The peptide linker gets cleaved by vitreal enzyme and the released drug reaches the target in retina and choroid. Aim of this thesis was to screen a library of 25 peptide linkers for cleavage in the presence of porcine vitreal enzymes. The peptide linkers were chemically synthesized and the in vitro stability of the peptide linkers were studied in freshly isolated porcine vitreous. Ten time point samples were collected over a period of 45 days and the peptide cleavage in porcine vitreous was assessed by LC-MS method. A TQ-S liquid chromatography-mass spectrometer was used to study the linker cleavage. LC-MS method development for the peptide library was carried out using IntelliStart wizard function. Out of the 25 peptide linker in the library, stability of eight linkers were not included in the LC-MS analysis as a mass method could not be developed. Out of 17 peptide linkers studied, 14 were categorized as fast cleaving linkers (>90% of the linker cleaved in porcine vitreous after 5 h). Three linker peptides; P4, P5 and P25 were categorized as slow cleaving linkers. Conjugating slow cleaving peptide linkers to small molecule drugs will increase the half-life and enhance the duration of drug action upon intravitreal injection. In this study, linkers that are hydrolyzed by specific enzymes present in vitreous or ocular tissues are exploited to investigate their potential for delivering small molecule drugs.

The solubility of a poorly water-soluble drug can be improved by converting the crystalline drug into an amorphous form. However, the amorphous form is metastable due to the higher energy state and recrystallization may occur during storage and dissolution. The amorphous form can be stabilized by forming an amorphous solid dispersion (ASD), where the drug molecules are dispersed to the solid medium, e.g. hydrophilic polymer. One preparation method for amorphous solid dispersions is spray drying, where a solution containing a drug and polymer is converted into small droplets in a drying chamber, in which the solvent evaporates in a hot gas stream and solid particles are formed. The aim of this study was to investigate whether an ASD of a poorly water-soluble drug can be prepared by spray drying using 20:80 (V/V) ethanol-water mixture as a solvent in a feed solution. Indomethacin (γ-polymorph) was used as a model drug and polyvinylpyrrolidone vinyl acetate (PVPVA) as a polymer. The aim was to find a suitable formulation where the drug is in the amorphous form after spray drying and remains in the amorphous form during storage. The ratios of the drug to polymer in the spray-dried formulations were 1:4, 1:6, 1:8, 1:10, 1:12 and 1:16. The study also examined whether a change in one process parameter, pump feed rate, affects the amorphous nature and stability of the resulting spray-dried solid dispersions. Two different pump feed rates, a higher 30% and a lower 15%, were used in the study. X-ray powder diffraction (XRPD) was used to characterize the solid-state of the spray-dried formulations. XRPD measurements were performed immediately after spray drying and on selected time points during storage. Formulations 1:10 at 30% feed rate, 1:12 at both feed rates and 1:16 at 30% feed rate were amorphous after spray drying. In 1:12 (30%) and 1:16 (30%) formulations indomethacin remained in amorphous form over the study periods (22 and 56 days, respectively). In other formulations, indomethacin was found to be in crystalline α-form immediately after spray drying or recrystallization to the α-form occurred during storage. The interaction between indomethacin and PVPVA was studied by surface plasmon resonance spectroscopy (SPR). The aim of the SPR measurements was to understand the interaction between these substances in the feed solution used in spray drying. PVPVA solutions of various concentrations (1%, 0.5%, 0.1% and 0.01%) were injected to the surface of the gold sensor coated with crystalline γ-indomethacin, and the changes in the SPR signal responses were monitored during the interaction. The same measurements were also performed on a pure gold sensor without indomethacin. An interaction between indomethacin and PVPVA can be observed, and based on the measurements, a polymer layer with a thickness of about 1 nm was formed on the surface of the indomethacin sensor regardless of the concentration of the polymer solution. Thus, even a small amount of polymer in solution is sufficient to cover the indomethacin crystals. This may also occur in the feed solution during spray drying, but further studies with SPR are still needed, especially with amorphous indomethacin. This study showed that an ASD of indomethacin and PVPVA can be successfully prepared by spray drying using an aqueous feed solution. Spray-dried 1:12 and 1:16 formulations at a higher pump feed rate were found to be stable enough for further studies. If the spray-dried material is further formulated into a pharmaceutical product, indomethacin must remain in amorphous form throughout the shelf-life of the product to maintain the improved solubility.

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.

Anticholinergic medicines are commonly used to treat e.g. incontinence. These medicines have side effects, which may cause and also exacerbate e.g. dryness of the mouth, increased heart rate, and even cognitive impairment. Older people may be more at risk for these side effects as they may be experiencing similar symptoms as a natural effect of aging, and because they may be using several medicines causing these effects. Older people often have a high medicine burden and also a high disease burden. Measuring anticholinergic effects to change medicine regimens and to reduce the symptoms is difficult as there is no golden standard method. This thesis investigated the published methods available for estimating anticholinergic burden in the literature review part, and used one anticholinergic scoring system, the Anticholinergic Risk Scale, in a cross-sectional study to test the effects of anticholinergics on mortality in 1004 older institutionalised patients from Helsinki area public hospitals. Cross-tabulations and Kruskal-Wallis or Chi square methods were used to detect differences between variables such as nutritional status or certain diagnoses when the patients were stratified according to their anticholinergic use. Cox Proportional Hazard regression, the logrank test and Kaplan-Meier curve were used to investigate the effects of anticholinergics on 5-year all-cause mortality. An in vitro serum assay and seven anticholinergic scoring systems were identified in the literature search. Also, 17 anticholinergic lists were identified, which covered 278 medicines, of which 21 appeared on at least eight of the lists. In the empirical study, the women's (n = 745) mean (± SD) age was 83.35 (± 9.99) years, and they were older than the men (n = 241, mean age ± SD 75.11 ± 11.48, p < 0.001). The 1004 patients (response rate 70 %) were using a mean (± SD) number of 7.1 ± 3.4 regular medicines (range 0-20). 455 patients used no anticholinergics, 363 had some anticholinergic burden (score 1 or 2), and 186 had a high burden, with anticholinergic scores of 3 or more. The mean ARS score (± SD) was 1.2 ± 1.5 (range 0-10). When three anticholinergic lists were compared, all three lists identified only 280/791 of patients who were anticholinergic users according to at least one list. No association was found between anticholinergic medicine use and mortality. There are several methods available for measuring anticholinergic burden, but there is a need for a consensus method. This was highlighted by the lack of agreement on medicines on different lists and when three anticholinergic lists tested identified different patients when compared to each other. Anticholinergic use was common in this frail, older patient sample, but no effect on mortality was shown in this study setting. The cross-sectional nature of the data limits the reliability of the study, and any conclusions beyond older patients in Helsinki area must be done very cautiously. Future research should define anticholinergics better and investigate their possible effect on mortality in a prospective, randomised, and controlled setting.

Multiple Sclerosis (MS) is an incurable autoimmune demyelinating disease affecting the central nervous system (CNS). Although the detailed pathogenesis remains unclear, recent research has highlighted the involvement of B cells. For decades, however, MS research was based on T cell-focused animal models of autoimmune encephalomyelitis (EAE), which do not reflect the involvement of B cells in the pathogenesis. Therefore, B cell-dependent EAE models are hypothesized to allow a better understanding of MS immunohistopathology and may therefore lead to the development of efficient treatments. In our spontaneous relapsing-remitting (RR) EAE model, B cells are recruited from the endogenous repertoire by transgenic myelin oligodendrocyte glycoprotein (MOG) -reactive T cells, causing the development of EAE in 3–4-month-old mice. Interestingly, MOG-specific antibodies are present long before actual onset of clinical disease and can be detected already in 5-week-old RR mice and disease development in RR mice is dependent both on the presence of (presumably MOG-specific) B cells as well as on stimuli provided by intestinal microbiota. Firstly, we evaluated the broader usability of induced germinal center cell (iGB) culture as a model for B cell repertoire studies. Then, by using iGB culture, we studied whether MOG-specific B cells are present in secondary lymphoid organs of younger than 4-week-old and germ-free RR mice. Finally, this study aimed to investigate whether the repertoire of MOG-specific B cells undergoes significant qualitative changes from young healthy mice to older acutely sick RR mice, and whether at the time of disease onset the recruited MOG-specific B cells expand and mature in the cervical nodes (cLN) or in the CNS. To do so, following the hosting-lab’s previous single-cell RNA sequencing (scRNA-seq) of B cells derived from cLN of 5-week-old RR mice, we performed the scRNA-seq of B cells from CNS, spleen, and cLN of acutely sick RR EAE mice. We demonstrated that iGB culture is an unsuitable tool to expand pre-activated B cells, and hence, in our hands it was inappropriate for repertoire studies. However, iGB culture proved to be useful for screening different organs for MOG-specific B lymphocytes, and we found that anti-MOG antibodies were firstly detected in 3-4-week-old RR mice, and MOG-specific B cells were present also in germ-free RR mice. Our scRNA-seq results revealed many highly expanded MOG-specific B cell clonotypes in acutely sick RR mice. Moreover, the B cell repertoire of sick RR mice was more diverse, including IgG1, IgM, IgG2b, IgG2c, and IgG3 isotypes, compared to healthy 5-week-old RR mice that had only IgG1 or IgM isotypes. Two-thirds of the expanded clonotypes were primarily detected in the CNS in sick RR mice, indicating that clonotypes develop further and continue isotype switching within the CNS. We also detected more somatic mutation in the variable region of expanded clones of sick RR mice compared to 5-week-old RR mice. The results of this study clearly show an antigen-driven evolution of the MOG-specific B cell repertoire from healthy young to acutely sick RR mice, which seems to occur mainly in CNS itself. In contrast, cLN are the major initial priming site of MOG-specific B cells in healthy RR mice, even under germ-free conditions. This suggests that commensal microbiota is not required for initial recruitment of MOG-specific B cells, but for the development of EAE. To further validate our encouraging scRNA-Seq results, it is necessary, in future experiments, to confirm the MOG-specificity of expanded clonotypes.

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.