Faculty of Pharmacy

 

Recent Submissions

  • Miettinen, Ilkka (Helsingin yliopisto, 2020)
    Treatment-resistant bacterial infections pose a major challenge to healthcare. In order to establish a chronic infection, bacteria must tolerate antimicrobial treatment and evade clearance by host immune system. The ability of bacteria to form biofilms, which are sessile communities of adherent microbes encapsulated in self-produced matrix of extracellular polymeric substances, contributes crucially to both of these traits. Biofilm bacteria produce numerous virulence factors that facilitate their adherence, invasion into host tissue, and evasion of the host immune system. Variable microenvironments within the biofilm give rise to metabolically sedentary subpopulations of bacteria, known as persisters, which can transiently tolerate antimicrobial chemotherapy. The biofilm matrix provides additional mechanical barrier against a selection of therapeutics. This thesis presents the optimization and validation of a Staphylococcus aureus model for persistence. Bacteria were monitored past the stationary phase transition to detect metabolic switching and a culture with halted energy metabolism and replication was confirmed to withstand extreme levofloxacin concentrations in a manner that was reversible by resurrecting growth. This model culture was used to study the effect of the metabolic status of the inoculum on biofilm characteristics. A label-free proteomics-based analysis of surface-associated proteins was carried out, and persister model-derived biofilms were shown to display boosted tolerance traits, such as oxidative stress defense and immune evasion, when compared to biofilms created from metabolically active cultures. This was reflected to increased survival in macrophage-like cells. A mixed biofilm model based on S. aureus and Pseudomonas aeruginosa, common co-habitants in chronic wounds and cystic fibrosis lung infections, was also established to study the effect of interspecies interaction on biofilm virulence and tolerance. A label-free proteomics approach was applied to concurrently compare surface-associated and extracellular protein profiles between mono- and co-cultured biofilms. This study provided proteomics-level insight that simultaneously covered multiple facets of virulence and tolerance in mixed-species biofilms. Several of the key findings here were found to be aligned with previously published functional studies. The proteomics studies also involved the compilation and improved annotation of S. aureus and P. aeruginosa theoretical proteomes, which are now fully accessible for future studies involving these or related bacterial strains. Altogether, this work demonstrates the applicability of label-free proteomics approaches in two different, clinically relevant settings involving bacterial biofilms. It also proposes models, methods, and proteomic workflows for the study of bacterial virulence and tolerance.  
  • Rosenholm, Marko (Helsingin yliopisto, 2020)
    Anesthetics are commonly used to induce unconsciousness and insensateness during surgery. However, the impacts of anesthetics on brain function go well beyond their acute pharmacological effects. Animal research suggests that the developing brain is particularly vulnerable to anesthesia, and even a single exposure may induce persistent neurobiological and behavioral consequences. Nevertheless, anesthetics have demonstrated remarkable therapeutic potential against some prevalent and debilitating brain disorders, especially major depression. Indeed, a single subanesthetic dose of ketamine has been reproducibly shown to alleviate depression and suicidal thinking within hours of administration, and the effects can last for days. Induction of brain-derived neurotrophic factor (BDNF) receptor TrkB signaling and synaptic plasticity have been intimately connected with ketamine’s antidepressant effects, but the precise mechanistic basis remains obscure. Notably, rapid antidepressant effects have also been reported with other anesthetics, including nitrous oxide (N2O) and isoflurane, and after somatic treatments such as electroconvulsive therapy (ECT) and sleep deprivation. In the first part of this thesis, we investigated the long-term behavioral effects of early postnatal exposure to repeated brief isoflurane anesthesia. We exposed mouse pups to anesthesia on three consecutive days at two distinct developmental stages, at postnatal days 7–9 or 15–17, and later tested the behavioral phenotype of the adult animals. Isoflurane anesthesia caused modest behavioral effects on locomotor activity and spatial learning and memory of the adult mice, depending on the age of the animals during the anesthesia exposures. In the second part, we investigated the effects of various anesthetics on depressive-like behavior and molecular signatures connected to the antidepressant effects of ketamine in rodents. We subjected rats to the chronic mild stress model of depression and subsequently exposed them to repeated brief isoflurane anesthesia for a total five times every three days. This administration regimen, however, was insufficient to normalize anhedonic behavior in the stressed rats. Cortical and hippocampal BDNF levels in these rats also remained unaltered. We then investigated the dose-dependent and temporal effects of different anesthetics on TrkB signaling, activity-dependent immediate-early genes (IEGs), and electroencephalographic (EEG) activity in mice. Here, we discovered that N2O upregulated several IEGs (markers of cortical excitation) during acute pharmacological effects, and that these effects were followed by a rebound emergence of EEG slow-wave activity (SWA) and TrkB signaling after treatment cessation. Similar concurrent upregulation of SWA and TrkB signaling was evident after a flurothyl-induced seizure (reminiscent of ECT) and during the effects of a sedative drug, medetomidine. Medetomidine, however, lacked antidepressant-like effects in the learned helplessness model of depression. This suggested that instead of only an increase in SWA and TrkB signaling, a preceding excitatory effect is also crucial for rapid antidepressant effects. This may also explain our observed lack of behavioral effects of deep isoflurane anesthesia. Moreover, even though ketamine’s antidepressant effects are associated with subanesthetic doses, we found that ketamine increases SWA and TrkB signaling, with the most pronounced effects observed at high anesthetic-sedative doses. These effects appear independent of hydroxynorketamine, an active metabolite of ketamine that has demonstrated antidepressant-like effects in rodents. In conclusion, we found subanesthetic ketamine, N2O, and flurothyl to induce SWA after their acute pharmacological effects subsided. Interestingly, this phenomenon resembles the well-known postictal (i.e., after seizure) slowing of EEG activity, which has been connected to the antidepressant effects of ECT. Furthermore, the emergence of SWA coincided with the upregulation of TrkB signaling. Based on our results, we propose that rapid-acting antidepressants induce two distinct phases in the brain, with an initial excitatory phase followed by a sedative-like brain state that is characterized by SWA and TrkB signaling. Further studies are needed to elucidate whether a similar phenomenon is shared by other treatments that have demonstrated rapid antidepressant effects (e.g., isoflurane, psilocybin). The current proposal provides a novel framework for future research that encourages expanding the research focus from the acute pharmacology of the treatments to homeostatic alterations that potentially emerge as an intrinsic response of the brain to a drug challenge.
  • Palomäki, Emmi (Helsingin yliopisto, 2020)
    Drugs must dissolve upon administration to have a therapeutic effect. Nowadays, most new drug candidates are poorly water-soluble, which makes this solubility issue a significant global challenge. Solubilization can be enhanced using formulation-based solutions, particle size reduction, salt formation, prodrugs or amorphization of the drug. This thesis concerns the last approach, amorphization. Unlike highly ordered crystalline materials, amorphous materials lack long range order. This leads to amorphous materials having greater molecular mobility and free energy, and consequently solubility, than their crystalline counterparts. However, the solubility benefits of the amorphous form come with a price, since the thermodynamic instability of amorphous materials means they tend to crystallize. Pharmaceutical products need to be sufficiently physically and chemically stable throughout their entire shelf life to ensure their efficacy and safety. In the case of amorphous drugs, there are still many aspects about crystallization that are not fully understood. The aim of the thesis was to investigate factors influencing the crystallization process in single and multiphase amorphous systems, as well as complexities in monitoring the progression of crystallization. The crystallization of several one- and two-phase amorphous systems were investigated, with the influence of both excipients and atmospheric gas on the crystallization process being investigated. Raman spectroscopy and X-ray diffractometry were used to monitor crystallization in the study, and their sensitivities and suitability to measure crystallization in the samples of interest were considered. Differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and optical microscopy were used to provide complementary information on crystallization processes. In the present thesis it was found that excipients and atmospheric gases that interact with amorphous material, even in the absence of mixing and specific interactions, can delay the onset of crystallization. Additionally, it was found that Raman spectroscopy is not necessarily suitable for crystallinity determination, when the opacity of the sample changes, as can happen at temperatures above Tg. Overall, this thesis demonstrates that several factors, beyond those usually considered in traditional single-phase solid dispersions, can influence crystallization, and that these, together with the effect of measurement technique artefacts, should be carefully considered when developing amorphous formulations.
  • Sinnemäki, Juha (Helsingin yliopisto, 2020)
    Polypharmacy, i.e. concomitant use of several drugs is common among older adults. This increases the risk of using drugs that are potentially inappropriate and harmful for geriatric patients. Automated dose dispensing (ADD) is a procedure that has been implemented in some European countries, particularly in the Nordic countries and the Netherlands to manage these risks in primary care. In the ADD service, regularly used medicines are machine-packed into unit-dose pouches according to administration times. The service is expected to enhance appropriate drug use and to prevent medication-related harm among older adults as well as to decrease medication costs, and save nurses’ working time in primary care. This doctoral study aimed to investigate the existing evidence on the outcomes of the ADD service, assess the service’s initiation process and evaluate its impact on drug use and quality. A systematic literature review was conducted to summarize the existing evidence on the outcomes of the service in primary care. The initiation process of the ADD service was investigated by surveying community pharmacies offering the service. The service’s impact on drug use and quality were investigated using a retrospective cohort study with matched controls applying nationwide register data. The literature was systematically reviewed until the end of 2019. 20 studies were included, and only two of them were controlled intervention studies exploring the outcomes of ADD in primary care. Consequently, the evidence for ADD’s impact on appropriateness and safety of medication use is limited, and lacking on economic outcomes. When the ADD service was initiated, the medication list was incomplete for more than half (63%) of the patients (n=147). Community pharmacists collected information on patient’s medication from multiple sources to reconcile the list. Some type of medication review was conducted for most (96%) of the patients when the ADD service was initiated for them. Most commonly (69% of the patients) it was a prescription review, which is the least comprehensive type of medication reviews. Medication-related therapeutic changes were implemented for almost half (43%) of the patients, and almost all (93%) had technical changes due to the ADD process requirements in their medications while initiating the service. The retrospective register-based controlled study revealed that ADD users (n=2073) had more starts and discontinuations in their medications compared to their matched controls (n=2073). The results also suggest that drug use was decreased after the ADD service was initiated. When the quality of drug use was assessed by explicit criteria for potentially inappropriate medications for older adults (PIMs by Beers criteria 2012), an improvement was found. However, more complex problems in the drug regimens could not be solved. When the quality of drug use was assessed with more complex criteria, such as concomitant use of three or more psychotropic drugs, the quality of drug regimens was not improved. The results of this study imply that medication reconciliation and review need to be integrated into the ADD service procedure as an essential part of it. Both information technology systems and processes in healthcare organisations need to be further developed to ensure that medication records and lists are up-to-date. More comprehensive medication review than prescription review needs to be implemented as a part of the ADD service procedure to ensure rational pharmacotherapy for the ADD users. When municipalities and healthcare providers are purchasing ADD services, medication reconciliation and review need to be included as part of the contract.
  • Gatta, Viviana (Helsingin yliopisto, 2020)
    To overcame the spread of bacterial resistance to traditional antbiotics, great interest has arisen towards antivirulence agents, compounds targeting virulence factors. In fact, as there is no link between growth and virulence, antivirulence agents are considered less prone to promote resistance development. In this context, quorum sensing (QS), a communication strategy among bacteria which regulates several bacterial functions including virulence, has been widely investigated for the development of QS inhibitors with the aim of limiting bacterial virulence. This study describes the development of a new assay for the discovery of inhibitors targeting LsrK, a key kinase for autoinducer 2 (AI-2) mediated QS establishment in enteric bacteria. LsrK in fact phosphorylates the AI-2 which, only in the phosphorylated form, can bind to the LsrR repressor and enhance the response to QS signals via activation of the lsr operon. The new assay was used for the screening of three different compound libraries. The best hits from the three campaigns were harpagoside and rosolic acid, presented in Study I, also active in cell-based AI-2 mediated QS assay. Additionally, the active compounds found in Study II and III provided interesting information about the catalytic site of LsrK. To facilitate the confirmation of hits selected by target-based assay and to offer a new tool for the rapid identification of QS inhibitors, Study IV describes the design, optimization and application of a new bioreporter strain, emitting luminescence as response to AI-2 mediated QS activation. The assay was used to test a set of 91 compounds selected to target the ATP binding site of LsrK. The same set of compounds was also tested in the target-based LsrK inhibition assay. The combined results led to the identification of 6 compounds, active in both assays, which thus may decrease response to QS by inhibiting LsrK. Additionally, 18 compounds were active only in the cell-based assay implying that they target other components of the pathway. These findings broaden our knowledge on LsrK and may be used as scaffolds to design compounds with improved properties. Furthermore, the AI-2 mediated QS interference assay represents an additional tool for the identification of QS inhibitors alone or in combination with target-based assays.
  • Parkkila, Petteri (Helsingin yliopisto, 2020)
    Lipids self-assemble into lipid bilayers, which divide bodily tissues into cells and into functionally specified compartments. Imbalances in the lipid composition and metabolism take part in severe neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. These conditions are currently only symptomatically treated, and the functional insight into the effects of the drugs in different stages of the conditions is lacking. In the field of pharmacy, the current drug design protocols rely on the separate evaluation of the binding affinity between target protein and drug and the extent of lipid bilayer permeation, which dictates how likely the drug is to reach its target. For example, the direct treatment of the central nervous system requires the drug to cross the blood-brain barrier. Most of the drug target proteins are, however, permanently attached to the lipid membranes. This thesis hypothesizes that lipids and proteins can act together in relation to drug action, which chemical variations in the membrane constituents can profoundly affect. Oxidative stress induces peroxidation of unsaturated fatty acids, modulating the membrane properties, such as the permeability to water. It is of importance to understand how lipid peroxidation influences the extent of segregation of the domains in the lipid membrane. Although the functionality of the domains \emph{in vivo} is elusive, they may be associated with a myriad of cellular functions, such as the attachment of the actin cytoskeleton. By measuring lateral diffusion of lipids in Langmuir monolayers, we showed that the presence of oxidized lipids could irreversibly modulate the miscibility of the segregated microscale domains. This may affect the action of proteins, drugs and other molecules that interact with such structures. Since biological membranes are incredibly complex, consisting of thousands of molecules, they are challenging to study. Therefore, model lipid membranes are used in membrane research. In this thesis, these model systems were characterized using label-free surface-sensitive analytical techniques. Using the membrane models, we showed that the membrane-bound catechol-\emph{O}-methyltransferase (COMT) is able to function at the membrane-water interface, suggesting that the membrane partitioning and orientation of its substrates and inhibitors influences the drug efficacy. Inhibition of membrane-bound COMT is desirable in Parkinson's disease since it elevates dopamine levels in the brain. Also, the inhibition prevents the methylation of levodopa, a dopamine precursor that is currently the primary therapeutic agent. Therefore, we studied the partitioning of dopamine and different catechol compounds to model lipid membranes. Partitioning to the membranes, where the existence of nanoscale domains is proposed, was limited. The partitioning also seemed to be modulated by the charge, lipophilicity and hydrogen-bonding capacity of the compounds and surface charge of the membrane. These factors also affect the orientation of the compounds in the lipid membrane, which can define the probability of the to-be-catalyzed moieties to reach the catalytic site of the protein. To conclude, the results of this thesis demonstrate that the lipid environment can modulate drug action, which may have consequences for the design of novel therapeutics for neuropathological conditions.
  • Itkonen, Jaakko (Helsingin yliopisto, 2020)
    Since their introduction in the late 20th century, therapeutic proteins have become an irreplaceable class of pharmaceuticals and are today used to treat a wide variety of diseases ranging from arthritis, diabetes, and various cancers to more recently, for example, asthma and migraine. In ophthalmology, the treatment of certain neurodegenerative diseases of the retina, such as age-related macular degeneration and diabetic retinopathy, has been revolutionized by therapeutic proteins that combat the pathological growth of abnormal blood vessels in the retina. As retinal diseases are some of the leading causes of vision loss and blindness globally, and expected to grow in prevalence with aging populations, the importance and need for such ophthalmologic therapeutic proteins is expected to increase. During the early development of therapeutic protein candidates, the production of functional protein in adequate amounts can often be a significant roadblock. In this thesis, the expression of soluble recombinant human ciliary neurotrophic factor (rhCNTF) – a neuroprotective protein with therapeutic potential against retinal neurodegeneration – in Escherichia coli was enhanced. Codon optimization of the hCNTF gene was combined with screening of different culture media, culture conditions, and fusion partners to pinpoint ideal expression conditions for rhCNTF. Following expression in the determined optimal conditions, the protein was purified with immobilized metal-ion affinity chromatography and gel filtration, and the in vitro activity of purified rhCNTF was demonstrated in a binding assay with its cognate receptor, CNTFRα. Overall, an 8–9 fold increase in soluble rhCNTF fraction and a 10–20 fold increase in yield was achieved, whereas earlier efforts to produce CNTF have commonly required purification from insoluble inclusion bodies and/or yielded low protein amounts. Furthermore, such a combinatorial approach is successful as a screening strategy for soluble expression and could be applied to other proteins of pharmaceutical interest. When taken out of their natural biological milieus, most proteins are only marginally stable and susceptible to environmental perturbations. As such, the formulation of a therapeutic protein aims to protect the protein and retain its stability and biological activity, and ultimately to guarantee the therapeutic efficacy and safety throughout the lifetime of the pharmaceutical. Here, further characterization, formulation, and stability studies were carried out with purified rhCNTF. The proper folding of purified rhCNTF was observed with circular dichroism spectroscopy and the biological activity of the protein was verified in a cell proliferation study with a CNTFα expressing cell-line. After screening, two buffers were chosen as storage buffers for rhCNTF. Whereas minute changes in rhCNTF’s oligomeric status were observed in only of these buffers, no changes in rhCNTF’s thermal stability were observed in either buffer during the study period. As such, these results provide a basis for further formulation development for rhCNTF. Although intravitreally injected therapeutic proteins have become the cornerstone in the management of retinal neovascularization, how and to what extent these and other proteins penetrate into the retina remains poorly understood. Here, permeation into the neural retina was observed with fluorescently labeled rhCNTF in ex vivo retinal explant models. Our results indicate that permeation to the CNTF-responsive target cells in the retina is not a limitation to exogenous CNTF’s direct neuroprotective actions. Moreover, our results provide further impetus to utilize ex vivo methods to systematically elucidate the retinal permeation and ocular pharmacokinetics of therapeutic proteins by and large. Therapeutic proteins have not only held the helm of best-selling pharmaceuticals for some time now, but currently also represent more than 40% of new pharmaceuticals in the development pipeline. Regardless, protein drug development has been plagued with ever increasing development costs yet with fewer new drugs entering the market, and there is an urgent call to disrupt this unsustainable cycle. While reasons for failure are diverse, for therapeutic proteins the most reported are poor therapeutic efficacy and immune responses, issues which are often encountered relatively late during the drug development workflow. Therefore, it would be of utmost utility to develop methods for detecting such susceptibility before significant effort and funds are spent in the development of less than ideal candidates. In this thesis, by integrating cell-free protein synthesis in small volumes together with split-intein mediated capture and light-triggered release, a streamlined platform for rapid protein production and screening was developed. Our results provide a proof-of-principle, with successful capture and release of protein of interest, as well as protein bioconjugation achieved using hCNTF as a model protein. The developed platform can be used for the rapid screening of therapeutic protein candidate producibility, and acts as a first module to be coupled to in-line assays for monitoring e.g. the candidate’s immunogenic potential, enabling such issues to be addressed/resolved already during early development stages.  
  • Toivo, Terhi (Helsingin yliopisto, 2020)
    Over the last decade, a great deal of research has described the medication safety risks in hospitals and institutional care both in Finland as well as globally. Less attention has been paid to the safety of medicine use in outpatient care, even though majority of the use occurs at home. The aim of this study was to enhance prospective medication risk management in outpatient care, by enhancing coordination of care with community pharmacists’ participation and use of risk management screening tools available. Specific objectives of studies I–III were: I) to demonstrate how community pharmacies can utilize their prospective surveillance system for screening clinically significant drug-drug interactions (DDIs) in outpatients and assess the rate of DDIs in a large national prescription sample. II) To integrate risk assessment tools, procedures and databases available in Finland to form a coordinated medication management model (CoMM) for older home clients involving home care nurses and practical nurses (PNs), physicians and community pharmacists. III) To assess the impact of the CoMM on medication risks identified in drug regimens of older home care clients over a one-year period. Medication risks assessed related to potentially inappropriate medications (PIMs), excessive use of psychotropics, anticholinergic and serotonergic load, as well as clinically significant DDIs. In study I, all DDI alerts issued by the online surveillance system were collected during a one-month period in 16 out of 17 University Pharmacy outlets in Finland, covering approximately 10% of the national outpatient prescription volume. The surveillance system was based on the FASS database, which categorizes DDIs into four classes (A–D) according to their clinical significance. Potential DDIs were analyzed for 276,891 dispensed prescriptions and they were associated with 11.2% of the prescriptions. Clinically significant DDIs categorized as FASS classes D (most severe, should be avoided) and C (clinically significant but controllable) were associated with 0.5% and 7.2% of the prescriptions, respectively. Studies II–III were conducted in primary care in the city of Lohja, Southern Finland. Health care units involved were the home care, public primary healthcare center and a private community pharmacy. System-based risk management theory and the action research method were applied to construct the collaborative procedure utilizing each profession’s existing resources in medication risk management of older (>65 years, n=191) home care clients. Study II produced a 5-stage medication management model (CoMM) suitable for screening medications of a high number of home care clients and identifying clients with potential clinically significant drug-related problems (DRPs). The core of the model was the triage meetings that proved to be a feasible method for customizing comprehensiveness of collaborative medication reviews, according to their clinical needs while minimizing physicians’ time demands. In study III, an RCT study design was used to assess the impact of the CoMM on medication risks identified in drug regimens of older home care clients over a one-year period. Participants’ (n=129) mean age was 82.8 years, 69.8% were female and mean number of prescription medicines in use was 13.1. The intervention did not show an impact on the medication risks between the original intervention group and the control group in the intention to treat analysis, but the per protocol analysis indicated a tendency for effectiveness, particularly in optimizing central nervous system medication use (benzodiazepines). Half (50.0%) of the participants with a potential need for medication changes, agreed on in the triage meeting, had none of the changes actually implemented. Study I demonstrated that community pharmacists can actively contribute to DDI risk management and systematically use their surveillance systems for identifying patients with clinically significant DDIs. In study II, the developed care coordination model (CoMM) was feasible for screening and reviewing medications of a high number of older home care clients in order to identify clients with severe DRPs and provide interventions to solve them, utilizing existing primary care resources. In study III, the CoMM intervention indicated a tendency for effectiveness when implemented as planned, particularly in optimizing CNS medication use during a 12-month follow-up. Our study revealed that organizations and health care units involved in home care clients’ medication therapy are currently working independently in silos, where no specific team membertakes holistic responsibility for medications. This study demonstrated the challenges to overcome when trying to change clinical practice and improve coordination between units involved in medication management of home care clients. Even though the outcomes of the intervention were not optimal, the value of the study is in discussing the real-world experiences and challenges of implementing new practices in home care. This study indicated that practitioners in Finnish health care are not well acquainted with systems thinking, a fact which needs to be addressed in the future. Further studies are needed on care culture and other contributing factors to high prevalence of PIM use and other risks for clinically significant DRPs identified in this study. Particularly, further investigation is needed on system-based factors contributing to situations where identified preventable clinically significant medication risks are left unsolved, as well as the relationship between inappropriate medication use and medication errors. A need for the organizational and national development of medication safety in primary care was identified in this thesis, which is line with the national and international publications, policy documents and recommendations. Furthermore, community pharmacists’ contribution to medication safety, particularly in older adults, should be better utilized in the future, as this thesis shows promising demonstrations. KEYWORDS: Medication risk management, medication-related risk, drug-drug interaction, primary care, home care, older adult, community pharmacy  
  • Sathyanarayanan, Gowtham (Helsingin yliopisto, 2020)
    Drug metabolism is a detoxification process by which the body converts pharmaceuticals into more hydrophilic metabolites. Understanding of the drug metabolism process and metabolic profiling plays a vital role in drug development processes by ensuring the safety and efficacy of treatments. Cytochromes P450 (CYP) are a superfamily of enzymes that are primarily responsible for metabolizing the majority of clinically relevant drugs. In preclinical drug development research, there is a constant need for the identification of metabolites and their CYP isoenzyme-specific elimination route, as well as possible drug-drug interactions thereof using high speed in vitro techniques. Miniaturization of the drug metabolism assays and related processes could further improve the throughput via parallelism and integration of several analytical steps on a single platform, as well as reducing the consumption of expensive reagents substantially. Micro total analysis systems (µTAS) usually refer to microfabricated devices that integrate several analytical unit operations, such as sample preparation, extraction, separation, and analysis on a single platform. These µTAS platforms can be either continuous-flow microchannel based systems or discrete droplet systems. Digital microfluidics (DMF) is one such technology, where sample droplets are manipulated individually on an array of electrodes. In DMF, the droplets of hundreds of nanolitres to a few microliters of volume can be dispensed, split, mixed, and merged independently via programmed and automated voltage application. In this thesis, several DMF-based bioanalytical concepts were developed and their feasibility for implementing droplet-scale drug metabolism assays was evaluated. In the first sub-project, droplet-scale immobilized enzyme reactors were developed by immobilizing CYP enzymes on porous polymer monoliths affixed onto a DMF platform. Assay incubation at physiological temperature was facilitated by localized heating of the DMF platform using integrated inkjet-printed microheaters. For the on-chip detection of drug metabolites, a protocol facilitating interfacing of the DMF device with a commercial wellplate reader was developed. In the second sub-project, the developed DMF platform, featuring the CYP reactors, were interfaced with ambient mass spectrometry (MS) via desorption atmospheric pressure photoionization (DAPPI). For in situ identification of the drug metabolites by DAPPI-MS, the chip design was optimized to be able to control the critical surface sensitive processes, such as sample precipitation and subsequent desorption/ionization directly from DMF surfaces. In addition, the feasibility of the same platform for a droplet-based liquid-liquid extraction of pharmaceuticals was demonstrated. All pharmaceuticals and metabolites analyzed could be detected with lower limits of detection in the range of a few picomoles. In the third sub-project, DMF droplet manipulation was interfaced with channel microfluidics to facilitate more versatile sample preparation such as separation of target analytes after the droplet-based enzyme reactions and prior to detection. To support the scaling up of the developed technology toward mass manufacturing, the entire device was assembled using low-cost inkjet printing and non-cleanroom polymer processing techniques. To achieve this interfacing, off-stoichiometric thiol-ene (OSTE) polymers were introduced as a new alternative dielectric material for the coating of inkjet-printed DMF electrode arrays, as well as for the integration of the microchannels with a DMF platform. In the fourth sub-project, magnetic bead based enzyme immobilization protocol was developed to facilitate screening the individual variation of CYP activities in donor-derived human liver microsomes (HLM) in droplet-scale. A CYP1A isoenzyme-specific model reaction was chosen to assess the inter-individual variation in the activities of this metabolic route in the liver microsomes collected from five individuals. The demonstrated protocol was shown to be technically feasible for biopsy-scale samples. In all, the new droplet-scale concepts developed in this thesis are first-in-their-kind examples of droplet-scale drug metabolism assays on DMF platform. The methods developed are generally qualitative or semi-quantitative and thus, in their present form, best feasible for the preliminary determination of metabolic clearance via CYP or identification of the produced metabolites of new drug candidates in vitro. Further development of the technology, particularly the enzyme immobilization process and the quantification of the produced metabolites, is needed to improve the wider applicability of the assays. It is noteworthy however that all of the fabrication processes and interfacing approaches taken in this thesis were carried out in regular, non-cleanroom laboratory conditions, which is foreseen to significantly improve the adaptability of the technology in any bioanalytical laboratories.
  • Lillsunde, Katja-Emilia (Helsingin yliopisto, 2020)
    Viruses are accountable for numerous diseases that form an immense threat to public health worldwide. The capability of viruses to continuously adapt to a changing environment makes the discovery of new treatments for viral diseases crucial. Natural products play an important role in the discovery of new drug candidates, essentially as a source of lead compounds that can be chemically optimized to achieve improved drug properties, such as safety and efficacy. In natural product drug discovery, the marine environment offers outstanding potential to discover new compounds with interesting bioactive properties. Marine species and their unique metabolites are still only partly discovered, and at the same time, vulnerable marine environments are threatened by human activities through pollution, overexploitation and climate change. The protection of marine biodiversity and sustainable use of marine resources is therefore a vital part of the study of marine organisms and their metabolites. This dissertation focuses on studying the antiviral properties of marine-derived compounds and their synthetic derivatives. The first part of this study covers screening of crude extracts from the Indian Ocean soft coral Sinularia kavarattiensis in a chikungunya virus replicon model followed by bioactivity-guided isolation and further study of the purified compounds. This study led to the isolation of six known norcembranoid compounds and the isolation and characterization of one novel compound, kavaranolide. Two of the isolated compounds were moderately active in the chikungunya replicon model, but also showed cytotoxic properties. The second part of the research focuses on studying the antiviral potential of synthetic compounds inspired by the marine sponge-derived alkaloids clathrodin and oroidin. In the screening of a compound library of 157 clathrodin and oroidin analogues in chikungunya virus and hepatitis C virus replicon models, four compounds were discovered to selectively inhibit the hepatitis C replicon with IC50-values ranging from 1.6 to 4.6 µM. Interaction with the cellular chaperone Hsp90 was proposed as the mechanism of action underlying the activity, and this hypothesis was supported by the results from molecular modelling and microscale thermophoresis interaction studies. Based on the study of clathrodin and oroidin analogues, 12 new compounds with a 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole structure were synthesized, in order to obtain improved binding to Hsp90 and improved antiviral properties. Three of the synthesized compounds showed improved binding to Hsp90 and specific inhibition in hepatitis C genotype 1b and 2a replicon models and moreover, inhibited the replication of full-length hepatitis C genotype 2a virus in a reporter virus RNA assay (IC50-values 0.03–0.6 µM). As Hsp90 is a host protein utilized by the viral replication machinery, antiviral activity achieved through inhibition of Hsp90 could be an attractive strategy to combat resistant viral strains.
  • Figueiredo, Patrícia (2020)
    Lignin is part of the lignocellulosic biomass and represents the second most abundant biopolymer after cellulose. However, only about 2% of the annually isolated lignin is used for low-value applications, mainly due to its complex structure. The fabrication of lignin nanoparticles offers a structural and morphological control of the lignin polymer, which enables lignin to be used for high-value products in biomedical applications like drug delivery and tissue engineering. Therefore, the main aim of this thesis was to exploit the potential of the under-investigated lignin-based nanoparticles as vehicles to deliver different therapeutic compounds for improved cancer therapy. Although multiple treatment options are available to treat cancer diseases, they still represent illnesses with very high incidence and mortality worldwide. Nanotechnology has opened doors to improve the limitations of current therapeutic modalities, such as chemotherapy or administration of immunomodulatory agents, by improving the solubility, stability and circulating half-life of the therapeutics, and by minimizing the systemic side effects. Firstly, different lignin-based nanoparticles were prepared and characterized, and their cytocompatibility investigated towards several cell lines. The ability of lignin nanoparticles to load different chemotherapeutic compounds was assessed, and drug release profiles were evaluated in two different buffers (pH 5.5 and 7.4). Secondly, after a carboxylation reaction of the original lignin polymer, the carboxylated lignin nanoparticles were surface modified with a polymer/peptide, as a proof-of-concept of the nanoparticles’ functionalization. The release profile of a poorly-water soluble cytotoxic compound (benzazulene) from the lignin nanoparticles was evaluated, and the in vitro antiproliferative effect investigated against several cell lines. Next, lignin polymers with different degrees of carboxylation were prepared in order to investigate the long-term stability of the resulting lignin nanoparticles at physiological conditions. After finding the optimal conditions in terms of stability, the lignin nanoparticles were further functionalized with a cell-penetrating peptide, and the cellular interactions of the resulting nanosystem were evaluated and compared with internalizing arginine-glycine-aspartic acid peptide-functionalized lignin nanoparticles, using two- and three-dimensional cell culture models. Finally, the lignin nanoparticles were loaded with resiquimod, an agonist of the toll-like receptors 7 and 8 that can induce the re-education of M2 to M1-like macrophages, and further functionalized with a peptide that targets the mannose receptor expressed by the M2 macrophages. Afterwards, the homing ability of these nanoparticles was investigated in vivo, using an orthotopic 4T1 triple-negative breast cancer model. The therapeutic effect of these nanosystems was studied in combination with a chemotherapeutic compound (vinblastine), and the immunological profile of the cells isolated from the tumors was compared. Overall, this thesis provides new insights on the use of lignin polymer as a novel starting material to develop lignin-based nanocarriers, in particular for cancer therapeutics.
  • Jumppanen, Mikael (Helsingin yliopisto, 2020)
    Cardiac disease continues to be a leading cause of death and hospitalizations in developed countries. Transcription factors GATA4 and NKX2-5 are master regulators of cardiac gene expression, taking part in multiple processes during heart development, as well as hypertrophy and recovery after e.g. myocardial infarction. Pathological hypertrophy is a homeostatic process, which often leads to cardiac dysfunction in pathological conditions (e.g. hypertension, genetic alterations, and myocardial infarction) further progressing to heart failure. There is an urgent need for treatments that would prevent disease progression at a molecular level. To date, no therapies have directly targeted the transcriptional regulation of cardiac hypertrophy. A novel treatment for this target would be particularly interesting, as current treatments are slowing the disease progression without directly targeting hypertrophic gene expression. The aim of this thesis was to design and synthesize nontoxic GATA4- NKX2-5 interaction inhibitors with antihypertrophic activity and determine their mechanism of action. Furthermore, the generated luciferase and toxicity assay data were analyzed to select compounds for further evaluation. In addition, different cycloaddition methods were explored for a facile synthesis of isoxazole scaffold. Finally, the mechanism of action of the original hit compound 1 was validated with affinity chromatography. In conclusion, novel inhibitors of GATA4-NKX2-5 transcriptional synergy were identified, which inhibit hypertrophic gene expression in rat cardiomyocytes. Remarkably, the immobilized hit compound (1) was shown to bind to GATA4 in the target validation study. In addition, with hierarchical clustering, a group of synergy inhibitors were identified that did not inhibit GATA4 transcriptional activity at 3 μM concentration. Further studies to determine the therapeutic potential of these more selective compounds are clearly needed.
  • Porokuokka, L. Lauriina (Helsingin yliopisto, 2020)
    Neurotrophic factor glial cell line-derived neurotrophic factor (GDNF), its co-receptor GDNF family receptor alpha 1 (GFRa1), and signaling receptor RET tyrosine kinase are essential to enteric nervous system (ENS) development; mice knockout for Gdnf, Gfra1 or Ret lack the whole ENS distal to the stomach. These Gdnf/Gfra1/Ret knockout mice die at birth because of lack of ENS and kidneys hindering analysis of postnatal function of those proteins. Transgenic overexpression in animal models on the other hand relates to loss of physiological spatiotemporal regulation of gene expression. These two bottlenecks have hindered the understanding of the role and therapeutic potential of GDNF/GFRa1/RET signaling in congenital diseases, such as Hirschsprung’s disease, and degenerative neurological diseases, such as Parkinson’sdisease. To tackle at least some of these problems, we have generated and characterized new mouse models with either increased or decreased gene expression dose - from the gene’s endogenous locus and limited to naturally expressing cells. Novel mouse models with increased expression were generated by editing 3’ untranslated region (3’UTR) of the Gdnf gene in such a way that the edited 3’UTR lacks binding sites for negative regulators such as microRNAs. By preventing the posttranscriptional downregulation via the 3’UTR we were able to achieve Gdnf overexpression from the endogenous locus limited to the naturally Gdnf expressing cells. We showed that 3’UTR replacement or 3’UTR editing results in increased GDNF levels in the brain and kidneys, maintaining the spatiotemporal expression pattern with positive effects on the dopaminergic system and negative effects on the kidney size and urogenital tract development. We also found that 3’UTR regulates GDNF levels in the gastrointestinal tract and that 3’UTR controlled GDNF levels determine proportions of neuronal subtypes in the ENS. More specifically, inactivation of negative Gdnf 3’UTR regulation enhances nitrergic and cholinergic neuron numbers, and leads to increased gastrointestinal transit time, increased stool pellet size, and increased stool water content. In congenital Hirschsprung’s disease (HSCR) patients, on the other hand, lack of ENS ganglia in the distal gut leads to constipation and megacolon. Even though RET mutations are the most common cause of Hirschsprung’s disease, no causative mutations in GFRa1 are known. However, one study reported low GFRa1 mRNA levels in some HSCR patients, suggesting that perhaps instead of being caused by mutations some HSCR cases could be triggered by reduced GFRa1 levels. Complicating the establishment of disease etiology in GDNF/GFRa1/RET related HSCR, postnatal viable HSCR mouse models with a defect in GDNF/GFRa1/RET signaling are not available. Here, we generated GFRa1 hypomorphic mice by insertion of a selectable marker gene in opposite transcriptional direction after the Gfra1 exon 6. Insertion of an expression cassette in the opposite transcriptional direction often leads to under-expression from the other strand, resulting in hypomorph allele. We showed that a 70-80 % reduction in GFRa1 levels in mice resulted in congenital Hirschsprung’s disease and associated enterocolitis phenotype with 100 % penetrance. We were also able to shed light in the chronology of events in the pathogenesis of Hirschsprung’s disease associated enterocolitis: first goblet cell dysplasia accompanied by an abnormal mucin phenotype is proceeding into epithelial damage, later followed by microbial enterocyte adherence and bacterial tissue invasion which likely leads to death by sepsis. Previously all those features had been described in patients but the sequence of events had remained unclear. Our results suggest that dysregulation of GDNF or GFRa1 levels by epigenetic mechanisms may play a role in normal and pathogenic development of the enteric nervous system.
  • Sipilä, Julius (Helsingin yliopisto, 2020)
    Phenolic compounds are ubiquitously encountered in all living organisms. To modulate the activities of endogenous and xenobiotic phenols, several families of Phase II metabolic enzymes have evolved, which can eliminate phenolic compounds through conjugation. In humans, the most important Phase II enzymes for phenol metabolism are UDP-glucuronosyltransferases (UGTs), cytosolic sulfotransferases (SULTs) and catechol O-methyltransferase (COMT). These enzymes increase the solubility of phenolic substrates, making them less active and easier to excrete. Because many clinically applied drugs also possess phenolic functionalities, UGTs, SULTs, and COMT are potentially important for the pharmacokinetics, exposure, and efficacy of therapeutics. In this thesis, the substrate specificity of human SULT1A3 and COMT were studied computationally, using comparative molecular field analysis (CoMFA). The CoMFA fields describe the shape, size, and electrostatic properties of the substrates, which are the most important determinants of molecular recognition by enzymes. In our models, variations in the substrate structures were statistically correlated with the changes in the enzyme kinetic parameters. In the SULT1A3 models, we found a clear preference for structural elements typically found in catecholamines. For COMT models, semi-empirical atomic partial charges were preferred over empirically parametrized charges. We added acid dissociation constant (pKa) values to improve the COMT models, and developed a modified Hammett equation to improve the pKa predictions for COMT ligands. As increasing amount of X-ray structural information has become publicly available, covering whole protein families, we attempted to extract relevant knowledge from the binding sites of several related proteins, to inspire ligand design. For this purpose, we created an automated data processing workflow, designed to process, combine, and analyze the electrostatic and knowledge-based contact preference fields of related proteins. This analysis was performed and validated using the nuclear receptor (NR) family of proteins and was later tested for the prediction of SULT isoenzyme substrates. In summary, the computational models that were developed in this study could be used in combination with other in silico approaches, especially MD simulations, to provide a better picture of the probable enzymes that may be relevant for the metabolism of a new phenolic drug or active metabolite. These models could be also used to design compounds with an improved affinity towards the studied enzymes, which may be clinically interesting due to the important roles played by SULT1A3 and COMT in the catecholamine-mediated neurotransmission pathways.
  • Esko, Terhi (Helsingin yliopisto, 2020)
    Societal Problem Solving and University Research Science-Society Interaction and Social Impact in the Educational and Social Sciences This study contributes to the understanding of social impact of research and its achievement in the educational and social sciences. The aim of the dissertation is to uncover how the interactions between researchers and their surroundings develop and how diverse fields in the educational and social sciences contribute to the society. In the context of innovation policy, which emerged in the 1990s and 2000s, the university has a central role in knowledge production. In the policy realm this was called the university’s third mission. Universities and researchers are expected to produce added value, innovations, and economic benefits for stakeholders outside the university, such as industry and political decision-makers. In addition to this, research is to contribute to complex societal and political questions. In innovation policy and the research evaluation literature, the focus has been on quantifiable outputs, which tend to favor the natural and technical sciences. In this dissertation, consisting of four articles, I follow the work and research findings of two research groups with the help of case studies. One of the cases focuses on the educational sciences and research on learning difficulties. The second case is an analysis of multidisciplinary urban studies and the study of social segregation. Both cases represent public good and policy-relevant research. The empirical data collection took place between 2011 and 2018 consisting of interviews, documentary data and policy guidelines. Several analytical strategies were used to ensure methodological triangulation of the data. The findings suggest that the social impact of academic research should be understood through its various dimensions: epistemic, artefactual, social-institutional and geographic. These dimensions depend on the context in which research is conducted but also on the stakeholders and beneficiaries that researchers have. In addition, the concepts of context and stakeholder should be analyzed with more nuance and detail in order to understand social impact. Keywords: social impact, academic research, universities, third mission, innovation, research evaluation, educational sciences, social sciences
  • Saari, Heikki (Helsingin yliopisto, 2020)
    Drug delivery aims to optimize the systemic distribution of administered drugs to reach their target tissue in an effective and specific manner. This approach is arising also in the field of cancer therapy, since traditional small molecule chemotherapeutics can cause severe side effects and more sophisticated biomolecular therapeutics cannot always reach the target cells on their own. For these purposes, therapeutics can be packed into carriers that improve their pharmacokinetics by providing a suitable, protected environment for the cargo to travel in the body, armed with targeting molecules that guide them to the site of interest. Synthetic drug carriers include liposomes, porous silica and different polymer particles that can be modified to contain necessary surface structures for targeted delivery and improved biocompatibility. While they have shown much promise in research settings, their success in cancer treatment has been limited to only a handful of commercially available formulations, some of which have presented liver toxicity as a result of continuous administration. Additionally, targeting cancer cells is difficult because cancer is a very individual disease, and heterogeneity exists even within tumors, so finding any universal cancer-specific markers to target is extremely challenging. Nature has adopted a similar approach for delivering molecules containing biologically functional cargo from cell to cell. These naturally occurring carriers are called extracellular vesicles (EVs), in contrast to intracellular vesicles that mediate cargo trafficking inside of the cells. EVs are 50 – 1000 nm in diameter, consisting of a lipid bilayer with embedded membrane proteins that encloses water-soluble biological cargo within. Their exact composition and cargo vary from cell to cell and depending on the condition of the cells that produce them. Additionally, cells have multiple pathways for EV secretion and production. For these reasons EVs comprise a very heterogeneous population of vesicles in composition and they also have multiple biological functions that have not been completely clarified yet. EVs have been found in practically all body fluids and organisms that have been studied, taking part in the normal and pathological functions of the organism. For example, procoagulant EVs are found in blood and saliva that participate in hemostasis, while EVs secreted by cancer cells promote the survival, growth and metastasis of the tumor. They are able to affect cells via membrane interactions and by delivering functional cargo into their recipient cells. It has been shown that EVs are even able to target cells selectively by binding to specific cellular receptors, enabling targeted cargo delivery. Given their natural cargo delivery properties, EVs present remarkable potential for drug delivery applications. As cancer cells also use EVs to communicate with each other, their EVs reflect the same heterogeneity that exists within the cells themselves, and may provide useful insights for cancer-targeted drug delivery. However, the internalization mechanisms of drug-carrying EVs and especially the fate of the drugs they carry is not well understood. In this thesis, the use of EVs was assessed for the delivery of chemotherapeutic drug paclitaxel and an oncolytic adenovirus that represent small molecular and biological chemotherapeutics, with cancer cell-derived EVs as the model carrier. The studies presented here focus on the preparation, characterization, intracellular tracking and effectiveness of these EVs for cancer therapy. The obtained results provide novel methods and knowledge for the future development of EV-based therapeutics for the treatment of cancer. First, EVs were loaded with paclitaxel by incubation, which enhanced the cytotoxic effect of the drug, changing its internalization from passive diffusion to endocytosis. A novel approach using fluorescence lifetime microscopy was introduced for tracking the release of paclitaxel from the EVs inside of the cells, identifying distinct patterns of subcellular drug release in individual cells and its intracellular kinetics. This method was able to show details about the drug release mediated by EVs that could not be observed with conventional fluorescence microscopy. Additionally, the relationship between EVs and adenoviruses was explored, revealing a previously undocumented pathway of spreading adenoviral infection via EVs. This EV-mediated infective delivery of the viral genome occurred separately from the classical pathway of adenoviral life cycle, and produced infective particles resembling EVs more than virions. EVs can in theory enhance the pharmacokinetics of oncolytic viruses by hiding them from the immune system and providing them alternative pathways for cell targeting and internalization, however it was found that infective EVs do react with adenovirus neutralizing antibodies. Taken together, these results suggest that EVs can act as versatile carriers of therapeutic cargo for cancer treatment, ranging from small molecule chemotherapeutics to oncolytic viruses, though they require further development. New methods are reported constantly for preparing and studying therapeutic EVs with new, innovative approaches that will help in the future treatments of cancer and other diseases.
  • Mononen, Niina (Helsingin yliopisto, 2020)
    Medicines information (MI) is an essential part of rational pharmacotherapy. Intensified clinical research and more matured pharmacovigilance systems have produced more information on therapeutic effects of pharmacotherapies to facilitate more detailed profiling of their benefits and risks. In turn, more open communication on medications with patients has been facilitated by drug safety issues, patients’ right to know about their treatments and by a significant increase in electronic information sources. Even though a wide variety of evidence-based MI sources for patients and consumers is currently available, the coordination between MI sources and their providers has been limited. The need for coordination has become more evident as the number of MI sources and providers has substantially increased over time. Improved communication on medicines to patients and consumers has been a strategic priority in developing MI practices in the European Union, including Finland, during the 2000s. To enhance the coordination of MI practices in Finland, the Finnish Medicines Agency Fimea published the first national MI strategy in 2012. The primary goal of the national MI strategy is to influence MI practices in all social and healthcare settings to reach the ultimate goal of well-informed patients who adhere to their medication. This thesis examines MI practices and policies in Finland during the 2000s. The primary goal of the thesis is to support the strategic development of MI and the implementation of the national MI strategy. The thesis comprises three independent studies (I–III) in which both quantitative and qualitative research methods were applied. They investigated development targets for MI practices in Finland based on a systematic review of the existing literature (I), assessed long-term trends in the receipt of MI among the Finnish adults (II), and evaluated how well the ultimate goal of the national MI strategy regarding well-informed adherent patients with chronic diseases had been achieved at the midpoint of the strategy period in 2015 (III). The systematic review on MI research conducted in Finland during 2000–2016 found 126 studies that covered a wide range of approaches applying various research methods (Study I). More than half of the studies were qualitative (54% of all studies, n=68), although surveys were the most commonly used individual method (47%, n=59). Twelve studies were interventions and only six studies applied a theory. Patient counselling in community pharmacies was the most commonly studied topic (19%, n=24). Regardless of some methodological pitfalls, MI research provides a multifaceted understanding of MI practices and their development needs in Finland. Research should shift towards larger research lines having a stronger theory base and study designs. Future research should be focused on the effectiveness of MI in different healthcare settings, along with the use of electronic MI sources and services, MI literacy, MI needs among patients and healthcare professionals (HCPs). Based on the nationally representative repeated postal survey “Health Behaviour and Health among the Finnish Adult Population” conducted by the National Institute for Health and Welfare during 1999–2014, physicians, community pharmacists and package leaflets were the main MI sources among adult medicine users aged 15–64 years (n=18862) throughout the study period (Study II). The use of the Internet as a MI source increased the most noticeably, being used by 1% of the adult medicine users in 1999 and 16% in 2014. The number of medicine users who did not receive MI from HCPs more than doubled (17% to 38%), and the number of medicine users who did not receive MI from any sources increased by sevenfold (4% to 28%) during the study period. It is necessary to continue research on trends in the receipt of MI at the population level and to identify population groups requiring special attention, such as senior citizens with multiple medications. Further evidence is also needed on factors contributing to a growing number of medicine users not receiving MI. According to the interviews among stakeholder representatives (n=79, 71%) involved in the implementation of the national MI strategy, the medication use processes for patients with chronic diseases requires development at every level of implementation (i.e., macro, meso, micro) (Study III). Medication counselling and other care advice by HCPs, particularly by community pharmacists, were the best implemented actions in general. The major actions needing development at the infrastructure level (macro) concern the coordination of care, transfer of patient information between care units, lack of reconciled medication lists, and local and national agreements on the responsibilities of patients and HCPs involved in the medication use process; at the HCP level (meso), focus on implementing the entire medication use process in primary and social care, particularly in geriatric units; and at patient level (micro), related to limited patient involvement in their care, lack of patients’ adherence to treatment and the inability of patients to retrieve information. Patients need to be better involved in implementing their treatment by improving empowerment and partnership to achieve the goal of well-informed adherent patients. KEYWORDS Medicines information, medicine user, patient, strategy, Finland
  • Anttila, Jenni (Helsingin yliopisto, 2020)
    Ischemic stroke is one of the leading causes of death and disability worldwide but the treatment options remain limited. Ischemic stroke, or cerebral infarct, occurs when blood flow to a focal brain region is restricted due to arterial blockage. Lack of oxygen and energy leads to rapid neuronal death in the ischemic region and to an inflammatory response via activation of brain-resident immune cells, microglia, and infiltration of peripheral leukocytes after ischemia-induced blood-brain barrier damage. Acute neuroprotective strategies need to be executed within a few hours after ischemia induction to be effective and have not proven successful in clinical trials. However, inflammation persists in the post-stroke brain and modulation of post-stroke inflammation could provide a therapeutic strategy with a large time window. Inflammation has both beneficial and harmful effects on injury progression but our understanding of many aspects of post-stroke inflammation remains incomplete. We characterized the neuroinflammatory response in the rat distal middle cerebral artery occlusion (dMCAo) model that was used to induce cortical infarcts in this thesis work. We found long-lasting inflammation and presence of phagocytic cells for up to 4 months after dMCAo, especially in the ipsilateral thalamus. We also found delayed neuronal loss occurring in the ipsilateral thalamus between 1-2 weeks after dMCAo due to connecting projection pathways between the cortex and the thalamus. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an 18 kDa endoplasmic reticulum luminal protein that is neuroprotective in experimental ischemic stroke models and has been associated with immunomodulatory properties. However, the knowledge of MANF’s recovery-promoting effects, mechanism of action, and endogenous expression pattern after cerebral ischemia are still limited. Thus, we characterized the endogenous MANF protein expression pattern in the dMCAo model and in ischemic stroke patient brains. Notably, we found that MANF protein expression is strongly induced in activated immune cells in the infarcted rodent and human brains. We also studied intracerebral post-stroke MANF therapy via viral delivery and recombinant protein injection and found that MANF promotes functional recovery when administered into the brain 2 days post-stroke as an adeno-associated viral (AAV) vector or as a recombinant protein starting 3-7 days post-stroke. Post-stroke MANF treatment did not alter the infarct size but the AAV-MANF therapy induced a transient increase in the number of phagocytic cells and innate immunity-related transcript levels in the peri-infarct area. In addition, we conducted a proof-of-concept study using intranasal MANF delivery to explore alternative delivery routes for administering the blood-brain barrier impermeable MANF protein. Pre-stroke intranasal MANF therapy decreased infarct volume and behavioral deficits. These data suggest a theoretical potential for intranasal MANF therapy, but bioavailability requires further improvement. As another approach, we investigated the efficacy of repeated post-stroke intranasal (+)-naloxone delivery in the dMCAo model. (-)-Naloxone is a small molecule drug which has been in clinical use for opioid overdose for decades and studied in the acute treatment of ischemic stroke because of its opioid receptor antagonizing effect. More recently, (-)-naloxone and its opioid receptor inactive (+) enantiomer have been shown to possess anti-inflammatory effects and to reduce microglial activation. (+)-Naloxone therapy, started one day post-stroke and continued for 7 days, decreased the infarct size and microglia/macrophage activation, and reduced behavioral deficits. This work broadens knowledge of the post-stroke neuroinflammation and secondary pathology of the thalamus in the cortical infarct model and shows for the first time that endogenous MANF protein is expressed in the activated, phagocytic immune cells in the infarcted human brain. This work also provides evidence on the recovery-promoting effects of post-stroke MANF and (+)-naloxone therapy and links both therapies with immunomodulatory functions.
  • Jokinen, Lenita (Helsingin yliopisto, 2020)
    The operating environment of community pharmacies is changing in Finland as a result of changes in society. Societal changes, such as municipal and service restructuring, and demographic changes, affect the operation of pharmacies. Pharmacies have expertise in implementing drug therapies that could be used more effectively as part of healthcare. Recent health and medicines policy developments in Finland allow pharmacies to expand their services and roles in healthcare. At the same time, the pharmacies' economy is influenced by past changes that have led pharmacies to streamline their operations and increase active product sales to secure their profitability. Little research has focused on the extent to which community pharmacy owners responsible for the operation and economy of pharmacies are committed to medicines policy. Pharmacy owners play a key role in influencing the strategic direction of the pharmacy business by deciding on their business as a private entrepreneur within the limits set by law. This doctoral dissertation explored the strategic work that guides the operation of community pharmacies, in particular the orientation towards health services and active product sales, and the factors involved. The study was carried out from the perspective of community pharmacy owners (studies 1-3) and key social and health care actors (study 4). The study was divided into four sub-studies. In 2013, data for the studies 1-3 were collected as a nationwide survey, targeting all private community pharmacy owners that were members of the Association of Finnish Pharmacies (n=581). The aim of the study 1 was to examine pharmacy owners' views on the strategic development of community pharmacy activities by 2020. The survey mapped the supply of pharmacy services at the time of the study (2013) and the pharmacy owners' views on future pharmacy services (2020). Two sum scales were compiled of the Likert-scale statements that examined the strategic orientation toward health services (13 statements, Cronbach's alpha 0.836) and active product sales (8 statements, Cronbach's alpha 0.699). The internal consistency of the sum scales was determined by using reliability analysis. The second study examined the strategy work of pharmacies and association between strategic planning and active product sales and healthcare service orientation. Responses were divided into two groups based on whether the responding pharmacy had a strategy or not. The relationship between strategy work and pharmacies' orientation towards health services and active product sales was investigated using the two sum scales created in the study 1. In addition, the relationship between strategy work and the background variables related to the pharmacy owner and pharmacy business was investigated. The third study investigated factors related to product sales and healthcare orientation in pharmacies using sum scales. Both sum scales were classified dichotomously by dividing pharmacies into either strongly or weakly oriented towards active drug sales, and strongly or weakly oriented towards health services. The relationship between the dichotomous sum scales obtained and the pharmacy background information and the actual pharmacy service offering was examined by cross-tabulation between the two variables. For the purposes of the analysis, the actual supply of pharmacy services at the time of the survey was also transformed into a sum scale consisting of ten services listed in the questionnaire. The aim of the fourth study was to determine the role and functional position of pharmacies in the future social and health service structure from the perspective of key social and health care actors. The data of study 4 were collected in late 2016 as a survey focused on the Ministry of Social Affairs and Health Steering Group for the Planning of Rational Pharmacotherapy Action Plan, including members, deputies and experts (n=149). A total of 198 pharmacy owners (34% response rate) responded to the nationwide survey. According to the responding pharmacy owners, the main task of community pharmacies was to operate as part of the healthcare service chain. The views were the same regardless of the characteristics of the pharmacy owner and their pharmacy business. Of the healthcare-oriented services available at the time of the study, the most common type of service was automated dose dispensing (77% of respondents' pharmacies). Of the pharmacies 21% provided comprehensive medication reviews (CMR). More than half (52%) of the responding pharmacy owners would have been prepared to increase the number of pharmacists with special qualifications (such as comprehensive medication reviews) if the service were to receive national quality criteria and funding. At the same time, the majority (90%) of respondents said they would invest in active additional sales, but few pharmacists (10%) saw their pharmacy primarily as a commercial company. In addition, almost all respondents (92%) fully or partially agreed that it is necessary for pharmacies to expand their product range to free-trade products in order to safeguard the pharmacy economy. Almost two-thirds (63%) of pharmacy owners reported they had a strategy. Strategy work (strategy/no strategy) had no influence on healthcare orientation but was linked to the pharmacy's active product sales orientation. Pharmacies where strategy work was carried out were clearly more likely to have active sales promotion than other pharmacies. The pharmacies that worked on the strategy were also interested in new services to ensure medication safety, such as the comprehensive medication reviews. Strategy work had no impact on other health care-oriented variables. The most common strategy work was in pharmacies with an owner of at least 10 years of pharmacy owner experience, a pharmacy with a large prescription volume and turnover. The actual provision of services by community pharmacies was investigated using sum scales. Of the ten potential services to be offered by pharmacies at the time of the study, 66% of pharmacy owners reported having up to two different services available at their pharmacy. The results showed a significant difference between the actual service provision and the intent of pharmacies. Pharmacy owners clearly saw the pharmacy as health-oriented and were interested in providing services, but in reality, very few pharmacies offered more than one or two services. According to this study, large pharmacies located in supermarkets and belonging to a marketing chain were clearly more focused on product sales than other pharmacies. Pharmacy owner and pharmacy-related background factors had no association with the healthcare orientation. The questionnaire sent to social and health care providers received 43 responses (29 % response rate). Respondents saw evidence-based self-medication counseling as one of the most important tasks of pharmacies (88 % of respondents). A significantly smaller proportion of respondents would be prepared to expand the range of non-prescription drug assortment (75 %). The most important services to be provided by pharmacies in the future were automated dose dispensing (93 %), medication reviews (82 %) and comprehensive medication reviews (80 %). According to the results of the study, pharmacy owners have a desire to develop healthcare-oriented services, but in practice, the activity has focused on active product sales to safeguard the pharmacy's economy. There is a big gap between willingness to provide services and actual delivery. The service offering is focused on services that can be implemented easily and with little investment, such as automated dose dispensing. Pharmacies should be supported through medicines policy in the development of services requiring larger and more long-term investments, which are of social and public health importance through the promotion of rational pharmacotherapy. Keywords: community pharmacies, pharmacy services, strategic development, healthcare, medicines policy, rational pharmacotherapy
  • Hanzlíková, Martina (Helsingin yliopisto, 2020)
    Gene therapy provides a promising option for treatment of various diseases, but the fact remains that the large number of gene delivery systems has met with little therapeutic success. Viral gene delivery has a high degree of specificity and efficacy, but it does not provide sufficient safety for clinical applications. Therefore, the search for an efficient alternative, a synthetic gene delivery vector, has been active. Typically, non-viral delivery vectors are based on the use of cationic polymers which bind and compact DNA via electrostatic interactions into nanoparticles (polyplexes). The ability of a cationic polymer to bind and condense DNA is important for effective delivery because good packing not only protects DNA against degradation in the extracellular space, but also allows effective release of DNA inside cells. While cationic polymers are relatively nontoxic and safe, they lack significant efficacy. This major drawback of non-viral vectors is largely due to a poor understanding of the mechanism underlying the complexation and gene delivery process. Furthermore, the lack of reliable methods to study the binding between DNA and cationic polymers has hindered development in synthetic gene delivery systems. The aim of this study was to investigate the mechanisms of DNA complex formation and gene transfer mediated by cationic polymers with different structures (poly-L-lysine, PLL; polyethylenimines, PEIs; poly-β-amino esters, PBAEs) and transfection efficiencies. This thesis combines time-resolved fluorescence spectroscopy with cell transfection studies in order to elucidate how polymer structure can affect DNA binding and influence gene delivery outcomes. This method allows the quantitative determination of polymer–DNA interaction and binding. We showed that the mechanism of PEI–DNA and PLL–DNA complex formation was positively cooperative with a saturation limit near 100% at a polymer/DNA molar (N/P) ratio of 2, whereas most of PBAE–DNA complexes expressed negative cooperativity and reached a saturation level close to 80%. The polymer topology, the type of amines (primary, secondary and tertiary) and their density, and the environmental pH had a clear effect on the binding constants and the degree of cooperativity. The possible correlation between fluorescence parameters and transfection efficiency was investigated with a series of PBAEs. Their transfection efficiency showed an increasing trend in association with the relative efficiency of PBAE–DNA nanoparticle formation. The role of free polymer in polyplex formation and gene delivery was examined with PEI as a model vector. For PEI polyplexes, the formation of the polyplex core was completed at N/P 2 and the excess of polymer formed a protective shell around the core. Unlike PLL, PEI molecules were able to undergo an exchange between the core and shell of the polyplexes. Such differences in structural dynamics of these polyplexes may partly provide an explanation for the differences seen in their DNA release and transfection efficacy at the cellular level. The excess of PEI in the shell had no effect on the physical state of polyplexes, suggesting that the polyplex core retains its original structure during shell formation. However, the excess of PEI was a crucial factor in successful transfection. The role of free PEI in the gene transfection process was examined in cell cultures with modified cell-surface glycosaminoglycans content. This study showed that free PEI is essential for minimizing the undesirable binding of polyplexes to cell-surface glycosaminoglycans, which may otherwise pose a barrier in non-viral gene delivery. Lastly, we focused on the role of PEI structure in PEI–liposome–DNA delivery systems (lipopolyplexes). We found that the enhancement of lipopolyplex-mediated delivery by different types of PEI species is common and associated with PEI size rather than structure. In conclusion, the present study demonstrated that the fluorescence spectroscopy approach for the analysis of gene delivery systems can provide valuable quantitative information about the binding behaviour of various cationic polymers to DNA. The improved understanding of mechanisms behind formation of these complexes can contribute to the design of polymeric delivery vectors with improved properties. Furthermore, this study sheds light on the mechanisms by which free polymer enhances gene transfer. It explains why high N/P ratios are needed for effective transfection and how the interactions between free polymer and cell-surface GAGs lead to alterations in gene transfer by the polyplexes.

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