Farmasian tiedekunta

 

Recent Submissions

  • 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.
  • Pöhö, Päivi (Helsingin yliopisto, 2020)
    Metabolites are small molecules present in a biological system that have multiple important biological functions. Changes in metabolite levels reflect genetic and environmental alterations and play a role in multiple diseases. Metabolomics is a discipline that aims to analyze all the small molecules in a biological system simultaneously. Since metabolites represent a diverse group of compounds with varying chemical and physical properties with a wide concentration range, metabolomic analysis is technically challenging. Due to its high sensitivity and selectivity, mass spectrometry coupled with chromatographic separation is the most commonly used analytical tool. Currently, there is no comprehensive universal analytical tool to detect all metabolites simultaneously and multiple methods are required. The aim of this study was to develop and apply mass spectrometry-based analytical methods for metabolomics studies. Neonatal rodents can fully regenerate their hearts after an injury. However, this regenerative capacity is lost within 7 days after birth. The molecular mechanism behind this phenomenon is unknown and understanding the biology behind this loss of regeneration capacity is necessary for the development of regeneration-inducing therapies. To investigate this mechanism, changes in mouse heart metabolite, protein, and transcript levels during the early postnatal period were studied. Non-targeted metabolomics methods utilizing liquid chromatography-mass spectrometry (LC-MS) and two-dimensional gas chromatography-mass spectrometry (GCxGC-MS) were applied to detect the metabolic changes of neonatal mouse hearts. Two complementary techniques increased metabolite coverage. A total of 151 identified metabolites showed differences in the neonatal period, reflecting changes in multiple metabolic pathways. The most significant changes observed in all levels (metabolite, protein, and transcript) were branched chain amino acid (BCAA) catabolism, fatty acid metabolism, and the mevalonate and ketogenesis pathways, thus revealing possible associations with regeneration capacity or regulation of the cardiomyocyte cell cycle. Insulin resistance (IR), metabolic syndrome, and type 2 diabetes have been shown to induce metabolic changes; the origin of the changes is unknown. In this study, human serum metabolite profiles from non-diabetic individuals were associated with IR. Gut microbiota were identified as a possible origin of the metabolic changes. Serum metabolites were detected with GCxGC-MS and lipids with LC-MS method. In total, 19 serum metabolite clusters were significantly associated with the IR phenotype, including 26 polar metabolites from five separate clusters and 367 lipids from 14 clusters. IR and changed metabolites were further associated with gut microbiota metagenomics and gut microbiota functional modules, showing that gut microbiota impacts the human serum metabolites associated with IR. Individuals with the IR phenotype had increased BCAA levels, which was influenced by bacterial species with increased BCAA biosynthesis potential and the absence of species with active bacterial inward BCAA transport. Sample throughput is often limited when chromatographic separation is used in metabolomics applications; a short analysis time is of great importance in large metabolic studies. The feasibility of direct infusion electrospray microchip MS (chip-MS) for global non-targeted metabolomics to detect metabolic differences between two cell types was studied and was compared to the more traditional LC-MS method. We observed that chip-MS was a rapid and simple method that allowed high sample throughput from small sample volumes. The chip-MS method was capable of separating cells based on their metabolic profiles and could detect changes of several metabolites. However, the selectivity of chip-MS was limited compared to LC-MS and chip-MS suffers more from ion suppression. Many biologically important low-abundance metabolites are not detectable with non-targeted metabolomics methods and separate more sensitive targeted methods are required. An in-house developed capillary photoionization (CPI) source was shown to have high ion transmission efficacy and high sensitivity towards non-polar compounds such as steroids. In this study, the CPI prototype was developed to increase its sensitivity. The feasibility of the ion source for the quantitative analysis of biological samples was studied by analyzing 18 endogenous steroids in urine with gas chromatography capillary photoionization tandem mass spectrometry (GC-CPI-MS/MS). The GC-CPI-MS/MS method showed good chromatographic resolution, acceptable linearity and repeatability, and low limits of detection (2-100 pg mL-1). In total, 15 steroids were quantified either as a free steroid or glucuronide conjugate from the human urine samples. Additionally, the applicability of the CPI interface for LC applications was explored for the first time using low flow rates. The feasibility of the LC-CPI-MS/MS for the quantitative analysis of four steroids was studied in terms of linearity, repeatability, and limits of detection. The method showed good quantitative performance and high sensitivity at a low femtomole level.
  • Bonabi, Ashkan (Helsingin yliopisto, 2020)
    A Bio-Micro-Electro-Mechanical-System (Bio-MEMS) is a miniaturized device that has mechanical, optical and/or electrical components for biomedical operations. High sensitivity, rapid response and integration capabilities are the main reasons for their attraction to researchers and adaptation of Bio-MEMS technology for many applications. Although the recent progress in microfabrication techniques has enabled a high degree of Bio-MEMS integration, many challenges remain. For example, extending the conventional cell monolayer cultures into 3D in vitro organ models often demands fabrication of round-cross sectional microstructures (microchannels and microwells) and integration of embedded metal-sensing elements. Owing to their low cost and the ease of the fabrication process, polymers have gained much attention in terms of biological microfluidic applications. Organically Modified Ceramics (ORMOCER) are hybrid inorganic-organic polymers, a new class of negative tone photoresist. Among polymers, ORMOCERs exhibit great potential with a view to biological microfluidic applications based on their inherent biocompatibility, transparency and mechanical stability. In this thesis, ORMOCER microfabrication methods were developed for implementation of optical, electrical and structural elements that are crucial for biological applications. A novel method, relying on controlled over-exposure of Ormocomp (a commercial formulation of ORMOCERs) was introduced for fabrication of tunable round cross-sectional microstructures, including microchannels (subprojects I-III) and microwells (subproject IV). Moreover, ORMOCER metallization was examined from the perspective of integration of embedded sensing elements (micromirrors and electrodes) into ORMOCER microfluidic channels to facilitate on-chip fluorescence (subprojects I and II) and electrochemical (subproject III) detection as well as electrical impedance spectroscopy (subproject IV). Metal adhesion, step coverage and bonding of embedded metal elements were addressed and new processes developed for various thin-film metals (subprojects III and IV). The round cross-sectional shape of the microchannel was exploited for implementation of thin-film reflective metal elements as concave micromirrors for optical detection of single cells, whereas the round shape of the microwells was applied to microfluidic three-dimensional (3D; spheroid) cell cultures. In addition to topography, the inherent surface properties of ORMOCERs were modified to allow for regulation of cell adhesion. As a result, cell monolayers (2D) and spheroids (3D) could be cultured side-by-side in a single microfluidic channel with non-invasive online impedance-based (monolayer) and optical monitoring (spheroids) of cell proliferation.
  • Novakovic, Dunja (2020)
    Active pharmaceutical ingredients can assume a diverse spectrum of solid-state forms. These forms differ in properties that are of utmost importance for pharmaceutical performance, such as solubility, dissolution rate and bioavailability. Thus, selection of a suitable solid-state form is highly important, particularly for the growing number of poorly water-soluble drug molecules. The amorphous (i.e. non-crystalline) form offers an often much needed solubility advantage at the price of thermodynamic physical instability and possible recrystallization. As the number of poorly water-soluble molecules in drug development is growing, so is the interest in the amorphous form and strategies for its stabilization. Formation of amorphous polymeric solid dispersions is the most common answer. However, issues with recrystallization, particularly at the surface, still remain, as well as high polymeric loadings. The importance of differentiating between the surface and bulk properties of pharmaceutical materials is becoming increasingly recognized. Processes such as dissolution or chemical reactions with surrounding materials start from surfaces. For drugs in the amorphous form, inevitable recrystallization starts at the surface. This transformed (partly) crystalline surface thus dictates dissolution (or lack thereof), as well as other properties relevant for manufacturing, shelf-life or administration. This is why understanding, monitoring and manipulating superficial phenomena is of such importance. Most established solid-state analytical methods show no or limited surface specificity. Nonlinear imaging (sum frequency generation (SFG) and coherent anti-Stokes Raman scattering (CARS)) are relatively new solid-state and surface specific methods. Moreover, as imaging methods, they can visualize solid-state distribution at the surface. Therefore, this thesis utilized novel nonlinear imaging approaches to better understand surface solid-state behavior. Further, the importance of surfaces in the crystallization, stabilization and dissolution of amorphous drugs was investigated. The first part of the thesis established surface and solid-state specific non-linear imaging methods capable of distinguishing multiple solid state forms. SFG imaging proved to be an excellent tool in detecting low levels of surface crystallization (undetectable with other analytical methods employed), in particular amorphous transformation to a single crystalline solid-state form. Additionally, multimodal nonlinear imaging (SFG and CARS modalities, each with their own benefits) was used for the first time in pharmaceutical samples to simultaneously differentiate up to three different solid-state forms. The second part of the thesis compared surface versus bulk crystallization and investigated surface crystallization (change in the solid-state form) during storage, as one of the key indicators of pharmaceutical performance. Both the cross-sectional SFG imaging of compressed powders, as well as the SEM morphology of continuous particle tablets allowed visualization of the surface-biased crystallization during storage. Further, the addition of different excipients physically mixed with the drug affected the crystallization of the amorphous drug in the bulk, however, their inability to stabilize the crystallization at the surface was demonstrated. In contrast, thin polymer coatings were successful in delaying the onset of surface crystallization at high humidity and elevated temperature during storage. The final part of the thesis investigated the implications of storage-induced surface crystallization and its stabilization with polymer coatings on pharmaceutical performance during dissolution, as a further necessary step in drug development. It was shown that different extents and natures of surface crystallinity affect drug dissolution. Multimodal nonlinear imaging revealed up to five solid-state forms simultaneously present at the surface, and aided the interpretation of the dissolution profiles. The initial dissolution rates of the short-term stored polymer coated samples were equivalent to those of the unaged uncoated samples. In summary, this thesis demonstrated the importance of surface solid-state properties, and their surface-specific analysis, for understanding the pharmaceutical performance of amorphous formulations during storage and dissolution. With further developments in amorphous drug formulations, the interest in surface crystallization and its prevention in the future will likely increase, together with the demand for surface-specific solid-state analysis. Altogether, it can be expected that in the future the understanding and utilization of surface phenomena will evolve from superficial to comprehensive.
  • Järvinen, Erkka (Helsingin yliopisto, 2019)
    Drug metabolism and transport are key areas in the drug development and therapy. The fate of a drug in the human body is determined by its physicochemical properties, which affect its absorption, distribution, metabolism and elimination processes within the body. The same processes also affect disposition of drug metabolites. Glucuronidation is the most important drug metabolism reaction besides oxidations. Glucuronidation of a drug produces glucuronic acid conjugates that are too hydrophilic to freely permeate cell membranes, and thus they require active transporters for their excretion. Knowledge of disposition of drug metabolites in humans is important for comprehensive understanding of the drug-related effects within the body. For example, glucuronide conjugates of some drugs inhibit drug-metabolizing enzymes and transporters, which causes drug-drug interactions in humans. Therefore, investigations of the molecular mechanisms of drug metabolite excretion are needed. Hepatic and intestinal ATP-dependent efflux transporters multidrug resistance-associated protein 2 (MRP2, ABCC2), MRP3 (ABCC3), MRP4 (ABCC4) and breast cancer resistance protein (BCRP, ACBG2) have been identified to transport glucuronide metabolites of drugs in animal experiments and human in vitro assays. However, systematic studies that compare the properties of these human transporters and characterize their transport kinetics are often lacking. In this thesis, the transport activity of human MRP2-MRP4 and BCRP was evaluated for 18 different glucuronide conjugates of drugs and drug-like compounds, such as androgens and estrogens. The major findings were that MRP2 and MRP3 are rather non-selective transporters and accept most of the glucuronides investigated as their substrates. MRP4 and BCRP, on the other hand, exhibit rather selective transport and these transporters were active only toward some of the glucuronides that were tested in this thesis. P-glycoprotein (P-gp, ABCB1), another important drug efflux transporter, was also included in the assays. However, this transporter did not transport any of the glucuronides investigated. Transport kinetic analyses revealed low Km values for MRP3, mostly clearly below 100 μM. This indicates that MRP3 is a high affinity transporter for glucuronide metabolites in the liver and intestine, where it is highly expressed. The Km values of MRP2 ranged from 120 to 800 μM. These values suggest that MRP2 is a low affinity, but possibly a high capacity, transporter in the same tissues as MRP3. MRP4 and BCRP exhibited Km values between 3-170 μM and 10-80 μM, respectively. In conclusion, MRP2, MRP3, MRP4 and BCRP are important efflux transporters that affect disposition of glucuronide metabolites of drugs in the human body. The affinity of glucuronides to these transporters may determine the different excretion of these drug metabolites in vivo, in either urine or bile.
  • Harjumäki, Riina (Helsingin yliopisto, 2019)
    There is an urgent need for better in vitro cell models to increase efficacy and cost-efficiency in drug development. Current simple models poorly mimic the natural in vivo cell environment. Human pluripotent stem cells (hPSCs) could serve as a limitless source for all the cells in the human body, but for most cell types, such as hepatocytes, efficient differentiation protocols do not exist. The signals that control cell behavior in vivo and in vitro are generated from growth factors (GFs), cell-extracellular matrix (ECM), and cell-cell interactions. The role of the ECM in cell behavior has only recently gained attention. Natural ECM of cells is a tissue-specific and complex three-dimensional (3D) array of various macromolecules. It provides physical, mechanical, and biochemical signals to cells. Mimicking the entire natural environment for cells is difficult, and it is, therefore, important to recognize the key components providing the essential signals. New materials, such as unmodified cellulose nanofibril (CNF) hydrogel, have been developed to tackle the technical difficulties that the ECM proteins have in 3D cell culture models, but the interactions of these materials with cells are not well known. Integrins with 18 subtypes are the main mediators of the cell – biomaterial interactions. The presentation and activation of these subtypes are important mediators in hPSC maintenance and differentiation. The activation of integrins can be caused by inside-out signaling through other integrins or receptors and outside-in activation through ECM molecules, divalent cations, or GFs. Hence it is vital to be able to measure these interactions in order to design good in vitro cell models. One of the most versatile instruments to quantify cell – biomaterial interactions and integrin activation is the atomic force microscope (AFM). The aim of this thesis is to study the hPSC interactions with biomaterials and use this information to better understand the cell behavior in vitro. The adhesion data of the AFM-based colloidal probe microscopy (CPM) correlate and predict cell adhesion on materials in vitro. Using CPM, we quantitatively tested the role of integrin density as well as integrin activation, enabled by cell viability and divalent cations, in these interactions. We observed that ECM proteins laminin-521 and laminin-511—detected in acellular matrix produced by hepatic progenitor cells—improved hPSC differentiation to hepatic cells. Cells in 3D cultures have more in vivo-like functions, and we, therefore, tested if the created differentiation protocol could be used to stepwise induce hPSCs specification to hepatic organoids in a CNF hydrogel. With CPM we found that CNF has only weak, nonspecific interactions with cells and maybe therefore CNF is not providing the signals needed for hPSC differentiation. The differentiation efficiency of hPSCs in CNF hydrogel is lower compared to matrix-free suspension culture. In conclusion, this thesis provides new quantitative information about cell – biomaterial interactions with a particular focus on hPSC cells, and laminin and CNF biomaterials. The implications of these interactions on in vitro cell cultures and stem cell differentiation to hepatic cells are analyzed.
  • Teppo, Jaakko (Helsingin yliopisto, 2020)
    Drug discovery and development, from basic research and drug design, through preclinical and clinical drug development, to approval of a new drug, is a lengthy and costly process, in which only a small fraction of the initial drug candidates make it to the market. The drug discovery pipeline consists of various types and methods of research, one of which is proteomics, the identification, quantification, and characterisation of proteins from biological samples. The objective of this work was to assess the feasibility of untargeted proteomics analysis in preclinical drug discovery and development, especially in combination with other omics techniques, by applying proteomics to three different subprojects in the field, each representing a different stage in drug research. In the first subproject, we studied the postnatal loss of regenerative capacity in the mouse heart, in order to identify potential drug targets for regenerative therapies. In line with the literature, we detected postnatal changes in energy metabolism and cell proliferation. In addition, we discovered that the ketogenesis and mevalonate pathways are temporarily activated in the mouse heart during the first week of life, and that ketogenesis plays a role in cardiomyocyte proliferation. These results highlight the importance of energy metabolism pathways as potential drug targets. The aim of the second subproject was to study the rat brain peri-infarct region during the recovery from ischemic stroke, and to elucidate how mesencephalic astrocyte-derived neurotrophic factor (MANF) exerts its neurorestorative effects known to induce functional recovery from stroke. We detected substantial stroke-induced changes in translation, lipid composition, and purine metabolism in the ischemic penumbra. The MANF-induced changes were limited to enhanced defence response to virus and decreased phagocyte infiltration. While insufficient for a conclusive mechanism of action, the results provide further evidence for the immunomodulatory effects of MANF. In the third subproject, we investigated the response of mouse pancreatic ductal adenocarcinoma (PDAC) tissue to treatment with the known drug tamoxifen. With proteomics validating the majority of the results from a series of other experiments, we discovered that tamoxifen induces widespread changes in the tissue architecture, involving increased vascularisation and decreased extracellular matrix (ECM) integrity. The effects are mediated by the G-protein coupled estrogen receptor (GPER). Finally, we showed that tamoxifen decreases proliferation and increases apoptosis, thus showing potentially beneficial effects in the treatment of PDAC. In conclusion, proteomics was successfully applied to each of the three subprojects to obtain meaningful data, but in all cases, data from other types of experiments was required to interpret and validate the findings. While global and comprehensive, abundance-based proteomics is biased towards structural and metabolic proteins, and the efficient research of e.g. signaling pathways would require the analysis of post-translational modifications, especially phosphorylation.
  • Nilsson, Sofia (Helsingin yliopisto, 2019)
    Understanding the mechanism of chemical reactions brings possibilities to optimization of reaction conditions. Microreactors coupled online to mass spectrometric detection provide a system highly suitable for mechanistic studies, enabling sensitive, selective, and rapid detection. By combining the information obtained with this experimental system with theoretical density functional theory investigations of the potential energy surface of the system, detailed information about the mechanism of a reaction can be obtained. On the other hand, molecularly imprinted polymers are useful tools for facilitating selective synthesis. This is achieved by formation of cavities within the polymer matrix, which are able to stabilize the transition state of the desired reaction. In this thesis, three different miniaturized reactors fabricated with additive manufacturing were combined online with electrospray ionization mass spectrometry for monitoring chemical reactions (Studies I-IV). The different miniaturized reactors were found to be variably suitable for this task. Overall, three different reactions were studied using miniaturized reactors coupled to a mass spectrometer – an inverse electron-demand Diels-Alder, followed by a retro Diels-Alder reaction (Studies I and II), an oxidation of a heptafulvene into the corresponding tropone by meta-chloroperoxybenzoic acid (Study III), and an acetylation reaction yielding the antibiotic drug linezolid (Study IV). The online mass spectrometry results obtained for the heptafulvene oxidation reaction were furthermore used as a basis for density functional theory studies of said reaction (Study III). Nine reaction pathways were investigated. The key step of the mechanism with the lowest energy barrier for oxidation of the studied heptafulvene into its corresponding tropone was identified as a Criegee-like rearrangement, while the overall reaction follows a Hock-like mechanism. Furthermore, highly porous molecularly imprinted polymer systems, which in flow injection quartz crystal microbalance studies exhibited enantioselectivity for a proposed transition state analogue of a transamination reaction, were developed and assessed (Study V). The molecularly imprinted systems prepared with n-heptane as porogen, and polystyrene beads, which, when extracted out, formed pores in the polymers that were imprinted with a molecule having either a D or L conformation of a proposed transition state analogue of a transaminase reaction, showed a clear selectivity for the transition state analogue enantiomer that they were imprinted with in flow injection quartz crystal microbalance studies. Otherwise these systems exhibited similar selectivity for the other analytes screened. The results presented in this thesis demonstrate that online combination of additively manufactured miniaturized reactors and mass spectrometry provides a convenient system for monitoring reactions online. At the same time, the results highlight limitations of the system such as memory effects arising from rough surfaces of the miniaturized reactors in combination with (from a mass spectrometry viewpoint) high concentrations of reactants used. However, the results from the oxidation study show that combinations of several methods can aid in overcoming limitations that one single approach may present. Finally, the developed hyperporous molecularly imprinted systems for enantioselective transamination reaction are promising for introduction into miniaturized reactors in the future.
  • Ollikainen, Elisa (Helsingin yliopisto, 2019)
    Drug metabolism is an important area of pharmaceutical research as it has significant effects on safety and efficacy of the therapy. Cytochrome P450 (CYP) enzymes metabolize the majority of clinically used drugs and have thus a critical role in their elimination process. Alterations in CYP activities can lead to unexpected adverse effects and toxicity (low activity), or on the other hand to complete lack of efficacy (high activity). Wide inter-individual variation in CYP activities is observed due to polymorphism as well as other individual and external factors. The emerging approach, called precision medicine, aims to increase the efficacy and safety of the treatment by considering the individual characteristic of the patient. The medication is then prescribed based on this information together with the diagnosis. Individual variation in CYP activities is one of the factors that should be considered when designing the treatment. Another aspect of precision medicine is targeted drug delivery with help of nanocarriers, which enables controlled release and accumulation of the drug at the targeted site. This approach improves the bioavailability of the drug and thus also the efficacy of the treatment, whereas side effects and toxicity can be decreased. Chemical analysis of a variety of different samples is involved in all areas of pharmaceutical research. Miniaturization of the analytical techniques results in fast and simple analysis of small sample volumes with reduced costs. Simple and portable miniaturized analytical decives have also enabled point-of-care analysis in e.g., doctor’s office. These techniques could provide valuable tools also for precision medicine and screening of the individual characteristics. However, the robustness, precision, and sensitivity of the microfluidic analytical devices should be further addressed before these techniques can compete with conventional methods in pharmaceutical research. The aim of this thesis was to evaluate the feasibility of microfluidic analytical techniques for pharmaceutical research. A particular emphasis was put on drug metabolism and its impacts on precision medicine. In the first subproject of this thesis, the effect of nanoformulations on CYP metabolism were determined in vitro. Three types of porous silicon (PSi) nanoparticles and three polymers commonly used in the same nanoformulations were investigated. Statistically significant alterations were observed in activities of the studied isoenzymes in the presence of the PSi nanoparticles, whereas polymers had less effect on the enzyme kinetic parameters. The highly polymorphic CYP2D6 was found to be most prone to inhibition by both the nanoparticles and the polymers. The results demonstrate the risk of interactions caused by other components of the (nano)formulations than the active ingredients. The effects of nanocarriers on CYP metabolism should be further investigated both in vitro and in vivo, to be able to evaluate the overall effects on CYP metabolism. In the second subproject, a paper microfluidic assay was developed for rapid screening of the inter-individual differences in CYP enzyme activities. The multiplexed microfluidic lateral flow assay was based on a paper-like functionalized calcium carbonate coating and inkjet printed hydrophobic fluid barriers. The assay was applied to study of individual differences in CYP2A6 and CYP1A2 activities in (human) liver microsomes (HLM) of individual donors. The determined CYP activities were compared to average activities in a 20-donor subpopulation. The results showed both increased and decreased enzyme activities in the HLM of individual donors compared to the pooled HLM. However, based on the comparison to in-solution assays, further validation of the microfluidic lateral flow assay is needed to reach the robustness and sensitivity required for routine use. In the third subproject, a commercial microchip electrophoresis (MCE) device with integrated electrochemical (EC) detection was applied to CYP metabolism studies and to analysis of morphine in mouse plasma and brain samples. The method developed for analysis of CYP metabolites showed good selectivity and precision. However, the sensitivity of the MCE-EC method was found insufficient for CYP metabolism studies. Instead, MCE-EC was shown to be feasible for quantitation of intraperitoneally administered morphine in mouse plasma and brain. Quantitation of morphine from biological samples was achieved with good precision and accuracy after off-chip liquid-liquid extraction (LLE) and on-chip electrokinetic stacking demonstrating the capability of MCE in targeted quantitative analysis. In the fourth subproject, MCE was combined with electrospray ionization-mass spectrometry (ESI-MS). The method was applied to separation of phosphorylated peptides, particularly the positional phosphorylation isomers, which is a challening task for conventional analytical techniques. The feasibility of the method was demosntrated with the help of monophosphorylated and triply phosphorylated insulin receptor peptides, which could be separated from the nonphosphorylated peptide in less than 40 s. The separation of the monophosphorylated peptide isomers from each other was achieved after derivatization. In conclusion, with help of selected applications, this thesis demonstrates the advances that microfluidics could provide for conventional pharmaceutical analysis and drug metabolism studies. MCE was shown to be suitable for quantitative analysis with good precision and selectivity. Sensitivity is a common challenge in the field of microfluidics, but with carefully selected method for each application, these techniques can reach the benefits of miniaturized analytical devices. Further improvements in integration of the sample pretreatment and enrichment on the same microchip would also enhance the sensitivity as well as decrease the variation associated with manual sample handling.
  • Huoponen, Saara (Helsingin yliopisto, 2019)
    Background: Rheumatoid arthritis (RA) and inflammatory bowel diseases (IBD), including Crohn´s disease (CD), ulcerative colitis (UC) and IBD unclassified, are chronic inflammatory disorders. In Finland, the prevalence of RA is estimated to be around 0.8% based on the data collected in the late 1980s, whereas the prevalence of IBD is around 0.9% in 2019. In the case of an insufficient response or intolerance to conventional drugs in the treatment of RA and IBD, biological drugs are a treatment option. Until May 2019, the European Medicines Agency (EMA) has approved ten biological drugs for the treatment of RA, four for CD, and four for UC. Biological drugs have proven to be an effective treatment for RA and IBD, and they have comparable efficacy and not significantly differing safety profiles. However, they are significantly more expensive than conventional drugs. Because of the high costs of original biological drugs, interest has grown in biosimilars and EMA has approved four biosimilars for the treatment of RA and two for IBD. RA and IBD, as chronic diseases, have a negative impact on patients’ lives, and, therefore, they decrease health-related quality of life (HRQoL). Furthermore, poorly treated RA and IBD may cause disability and uncontrollable costs for social and health care. Objectives: The aim of this study was to evaluate the costs, effectiveness, and cost-effectiveness of biological drugs in the treatment of RA and IBD. Methods: Systematic literature reviews (SLRs) were performed to identify published data on the cost-effectiveness of biological drugs for RA and IBD (study I and II). The SLRs were performed following current recommendations for SLR of economic evaluations to improve the quality and reliability of the study. A patient-level simulation model (study III) was developed to predict costs and outcomes associated with four biological drugs (abatacept, tocilizumab, rituximab and Tumour Necrosis Factor Alpha (TNF) inhibitors) in the treatment of RA patients who have previously been treated with TNF inhibitors. Following lack of efficacy or adverse events, the patients were switched to another biological drug until all four options were exhausted. The patients’ baseline characteristics and regression models used in the simulation were based on observational data from the National Register for Biological Treatments for RA patients in Finland. Several subgroup and deterministic sensitivity analyses were conducted. In the single-centre prospective observational study (IV), all IBD patients receiving maintenance infliximab therapy at Helsinki University Hospital (HUS) were switched to biosimilar infliximab (CT-P13). HRQoL was measured using the generic 15D utility measurement and the disease-specific Inflammatory Bowel Disease Questionnaire (IBDQ). Crohn´s Disease Activity Index (CDAI) or Partial Mayo Score (pMayo), and faecal calprotectin (FC) served for evaluation of disease activity. Data were collected at time of switching and at 3 and 12 months after switching. Patients´ characteristics and clinical background information were collected from patient records and costs were obtained from the clinical patient administration database of the hospital. Results: The SRL (I) of the cost-effectiveness of biological drugs for the treatment of RA showed that biological drugs did not seem to be cost-effective among conventional synthetic disease-modifying anti-rheumatic drug (csDMARD) naïve or csDMARD resistant RA patients with the cost-effectiveness threshold of 35000 €/QALY (Quality-Adjusted Life Year), but they might be cost-effective among csDMARD resistant patients with the threshold of 50,000-100,000 €/QALY. Rituximab was the only biological drug that seemed to be cost-effective among RA patients with a previous exposure to TNF inhibitors. According to the patient-level simulation model (III), drug costs were the lowest for rituximab in RA patients who had been previously been treated with TNF inhibitors, but when administration costs and costs of switching were included, drug costs were the lowest for TNF inhibitors. Abatacept was associated with the highest drug costs, whereas rituximab was associated with the highest outpatient and inpatient care costs. In total, TNF inhibitors had the lowest and rituximab the highest direct costs (including drug costs, administration costs, costs of switching, outpatient and inpatient care costs). The amount of QALY gained ranged from 9.41 for rituximab to 9.66 for TNF inhibitors. TNF inhibitors, abatacept, and tocilizumab had lower costs and higher QALYs than rituximab, and were therefore dominant in comparison to rituximab. According to the SLR (II), biological drugs seemed to be cost-effective for the treatment of active severe IBD with the cost-effectiveness threshold of 35,000 €/QALY, but the cost-effectiveness remained unclear in the maintenance treatment. Based on the prospective observational study (IV), no statistically significant difference was observed over one year following switching to the IFX biosimilar when the generic 15D instrument for the measurement of HRQoL in IBD patients was used. HRQoL measured with the IBDQ was, in CD patients, statistically significantly better (p=0.018) 3 months after switching to the infliximab biosimilar than at time of switching. Statistically significant finding was not observed in UC patients. Disease activity, in light of CDAI, pMayo and FC, was similar over one year following switching in IBD patients. The costs of biosimilar infliximab were around one third of the costs of originator one, whereas costs related to secondary healthcare (excluding the costs of infliximab) were similar before and after switching to biosimilar. Conclusions: The patient-level simulation model based on Finnish real-world data showed that TNF inhibitors, abatacept, and tocilizumab were dominant in comparison to rituximab in RA patients, who had been previously been treated with TNF inhibitors. Significant differences were not observed in effectiveness between biological drugs. As TNF inhibitors had the lowest costs and highest QALYs, so they were the most cost-effective treatment option. In contrast to the results of patient-level simulation model, rituximab was the most cost-effective biological drug among RA patients with an adequate response to TNF inhibitors based on SLR. The systematic search of the literature revealed that biological drugs seemed to be cost-effective for the treatment of active and severe IBD. Based on the Finnish observational data, it suggested that HRQoL and disease activity of the infliximab-biosimilar were comparable to the originator one in the maintenance treatment of IBD. The costs of the infliximab-biosimilar were significantly lower than the costs of the originator one, and switching from originator infliximab to a biosimilar one had no effect on costs related to secondary healthcare (excluding the costs of infliximab).
  • Uhari-Väänänen, Johanna (Helsingin yliopisto, 2019)
    Ethanol use disorders affect a vast number of people worldwide. In some individuals, controlled ethanol intake can gradually progress via ethanol abuse into addiction, characterized by escalated, uncontrolled and compulsive ethanol seeking and intake despite its negative consequences. A negative emotional state is common when ethanol is not available. The relapsing nature of this chronic disease also makes it difficult to treat. As the clinical efficiency of the currently available pharmacotherapies is relatively low, new treatment strategies are needed. The µ- and κ-opioidergic systems interacting with the brain’s reward pathway have been suggested to be central in controlling ethanol intake. The µ-opioidergic system is attributed to the rewarding and positive reinforcing effects of ethanol while the κ-opioidergic system is attributed to its negative reinforcing effects. It has been suggested that the µ-opioidergic system is more important in controlling ethanol intake while intake is still under control, while the role of the κ-opioidergic system increases as ethanol intake becomes more chronic, compulsive and relapsing. The main aim of this thesis was to clarify the role of µ- and κ-opioidergic mechanisms in the nucleus accumbens shell, a main brain area of the reward pathway, in controlling intermittent and relapse-like ethanol intake in rats. The used paradigms can roughly be considered to model aspects of ethanol intake before and after addiction has developed. Selective µ- or κ-opioid receptor agonists and antagonists were administered locally into the nucleus accumbens shell and systemic injections were used to elucidate this brain area’s overall role in controlling ethanol intake. These studies were undertaken as there is a gap in the knowledge on how the µ- and κ-opioidergic systems interacting with the nucleus accumbens shell affect ethanol intake and addiction-related behaviors per se. A high innate µ-opioidergic tone in the nucleus accumbens shell of alcohol-preferring Alko Alcohol (AA) rats has been proposed to account for their high ethanol preference but pharmacological studies are lacking. As local infusions of a selective µ-opioid receptor antagonist increased and agonist tended to decrease intermittent ethanol intake, the results support the notion that nucleus accumbens shell µ-opioidergic mechanisms participate in controlling ethanol intake and reward in AA rats. The role of nucleus accumbens shell κ-opioidergic mechanisms in controlling intermittent ethanol intake has not been extensively studied. Intra-accumbens shell administration of a selective κ-opioid receptor agonist had no effect but JDTic, a selective κ-opioid receptor antagonist, showed a weak long-term ethanol intake decreasing effect in AA rats. When these results are combined with the long-term decreasing effects shown after systemic JDTic administration, the results suggest that κ-opioid receptors are indeed able to control intermittent ethanol intake and the nucleus accumbens shell is one site participating in mediating these effects. The effects of JDTic on relapse-like ethanol intake in Long-Evans rats was examined because of the positive results from the previous study, earlier reports suggesting an increased tone of the accumbal κ-opioidergic system as ethanol addiction evolves and the lack of knowledge on what role the nucleus accumbens shell κ-opioid receptors have in relapse to ethanol intake. Both intra-accumbens shell and systemic JDTic attenuated relapse-like ethanol intake. These results suggest that the κ-opioidergic system interacting at least with the nucleus accumbens shell participates in controlling relapse-like ethanol intake. As the reference drug naltrexone, a non-selective antagonist, administered systemically also inhibited relapse-like ethanol intake, µ- and possibly also δ-opioidergic systems seem to have a role in mediating relapse. Taken together, these findings suggest that µ- and κ-opioidergic mechanisms are important in controlling intermittent ethanol intake and relapse to ethanol intake and the nucleus accumbens shell is one anatomical site mediating these effects. The results also suggest that selective κ-opioid receptor antagonism could be a feasible treatment strategy for ethanol use disorders.
  • Rinne, Maiju (Helsingin yliopisto, 2019)
    The diversity of G protein-coupled receptors is a fundamental element in this thesis. GPCRs are the largest family of membrane proteins, with about 800 in humans. While the human GPCR repertoire is well described, in other species, especially in non-mammals, GPCRs are not tracked to the individual subtype level in large-scale genomic studies. The diversity of GPCRs in non-human vertebrates was studied in the first publication. The study classified 142 rhodopsin-like non-olfactory GPCRs without human orthologue, 69 of which were reported for the first time. The study also points out inconsistencies in the GPCR nomenclature system and reveals a pool of yet-to-be studied receptors. Understanding the repertoire of GPCRs in non-human species might also be useful for many areas of science, such as pharmacology, ecotoxicology and evolutionary biology. For more insight into the orexin system, two small-molecule orexin receptor agonists were pharmacologically characterised in the second and third publications. Nag26 was confirmed to be a potent and almost full agonist of orexin receptors, but the selectivity for orexin receptor type-2 was not as strong as reported (20-fold against 70-fold). Yan7874 was also confirmed to be an orexin receptor agonist, but only partial and weak with high off-target activity. Neither of these compounds is likely to be suitable for further drug development as such. These studies also display the challenge in the development of small-molecule agonists for peptide-bound GPCRs. Finally, Ciona intestinalis putative orexin receptor was studied, and its functionality was verified in recombinant cells. Homology models of C. intestinalis orexin receptor revealed a highly similar binding cavity as in the human OX2 orexin receptor. Thus, the functionality of the receptor was studied in Ca2+ elevation assay, and the receptor was verified to be functional and binding to human orexin peptides. Further database mining resulted in the identification of putative C. intestinalis prepro-orexin and orexin peptide that was shown to bind to a plasma membrane of C. intestinalis orexin receptor-expressing cells. Solving the function of the orexin system in distinct species such as C. intestinalis might be interesting from an evolutionary point of view but also essential in extending the knowledge of the orexin system in humans. This thesis was conducted collaboratively with the Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy and Department of Veterinary Biosciences, Faculty of Veterinary Medicine, at the University of Helsinki.
  • Mgbeahuruike, Eunice Ego (Hansaprint, 2019)
    Piper guineense is a medicinal plant that has wide application in African traditional medicine where it is often used in the treatment of bacterial and fungal infections. It is an economic plant with numerous health benefits which is also consumed regularly as a functional food. The fruits, leaves and seeds are used as spices and flavouring agents in commercial food preparations in West Africa. The extracts are also used for the treatment of various diseases ranging from diarrhea, intestinal diseases, rheumatoid arthritis, bronchitis, cough, stomach ache, asthma to febrile convulsions, fever and mental disorders. There is also recent interest on the biological and pharmacological properties of its bioactive compounds such as piperine, the main alkaloid constituents of P. guineense which is responsible for its pungent aroma. Based on these numerous ethnobotanical, traditional and economic uses of this plant, it became interesting to evaluate the bioactive compounds present in the extracts and to further screen the extracts against some selected human pathogenic bacterial and fungal strains so as to ascertain the efficacy of the extracts and its compounds as potent antibacterial and antifungal lead compounds. Microbial resistance to the currently available antibiotics is a global problem that has resulted to a constant search for a new antimicrobial drug with strong efficacy and low cost. There is need to screen the extracts and bioactive compounds from P. guineense for possible lead compounds for antibacterial and antifungal drug discovery. In this study, first, a method was developed for the chemical profiling, qualitative and quantitative analysis of P. guineense extracts and a good mobile phase composition was developed for the high performance liquid chromatography (HPLC) and thin layer chromatographic (TLC) analysis of the extracts. The effect of the chamber type on the separation was also evaluated using unsaturated horizontal chamber in sandwich configuration, horizontal chamber in non-sandwich configuration and twin-trough vertical chamber. Furthermore, the in vitro antibacterial activity of the extracts were evaluated using 8 pathogenic Gram-positive and Gram-negative bacterial strains. An ethnobotanical survey was also conducted on the use of P. guineense extracts in the treatment of fungal infections in West African traditional medicine. The study area was chosen to be Imo state, South Eastern Nigeria were P. guineense is mostly used by traditional healers for the treatment of fungal infections which is often common among those suffering from HIV and AIDS. The aim of the survey was to document the various methods of preparations and administrations of these extracts for the treatment of fungal diseases. From this ethnobotanical approach, the leaves and fruits extracts of the plant was further tested against 5 fungal strains including Cryptococcus neoformans which causes meningitis in immunocompromised individuals. HPLC and TLC methods were developed for the analysis of P. guineense extracts with emphasis on the shortest analysis time and minimal solvent consumption, and the best mobile phase giving favourable resolution of bands was found to be toluene: ethyl acetate (PS 6-4 corresponding to 60:40 % v/v). The result of the TLC analysis showed that the developing chamber conditions does not affect the TLC separation efficacy in the analysis of P. guineense extracts. The extracts were active against the tested bacterial and fungal strains with minimum inhibitory concentration (MIC) values ranging from 19 to 2500 µg/mL.
  • Liu, Zehua (Helsingin yliopisto, 2019)
    Considerable efforts have been made to fabricate nano-sized drug delivery systems (DDS) with unique and advanced features in comparison to conventional DDS. Yet, challenges still lay ahead requesting for more controllable, even on-demand drug release profiles from the DDS. Moreover, the emerging concept of personalized treatment further urges the combining of therapy and imaging regimes into a single nanocarrier. Among all the nanomaterials studied so far, porous silicon (PSi) draws increasing interest for constructing DDS due to its good biocompatibility, non-immunogenicity, large pore size/surface area and easily changeable surface properties. Herein, the aim of this thesis was to explore PSi-based DDS for multiple biomedical applications, which were designed and synthesized with specific on-demand features. Moreover, simultaneous incorporation of imaging modalities and drugs enables real-time visualization of drug release and/or cellular/tissue level disease condition, which are expected to be beneficial for personalized treatment regime. First, the potential of PSi nanoparticles for hydrophilic drug loading and on-demand release were evaluated by adapting a dynamic non-covalent bonding method. Different ligands were synthesized and applied for modifying the PSi, and the hydrophilic anti-cancer drug doxorubicin (DOX) was sequentially loaded into the fabricated DDS for pH-responsive release profiles. Meanwhile, the fluorescence spectrum of DOX can be dynamically shifted or quenched, depending on the loading and releasing process, thus facilitating the in situ visualization of the drug releasing process. For hydrophobic drugs, a physical encapsulation method was applied to seal the pores of the PSi by a polymeric matrix. Microfluidic-assisted nanoprecipitation method was applied to synthesize batches of nanohybrids with identical PSi-core/polymer-shell structures, and the release behavior was feasibly tailored by the degradation behavior of the outer polymeric matrix. The first trial was set to fabricate a core/shell nanohybrid, with PSi and gold nanoparticles co-encapsulated in a pH-responsive polymer to simultaneously deliver hydrophobic drug and increase the computed tomography signal for acute liver failure theranostics. The newly established single-step co-encapsulation of different particles endowed a system with multi-functionalities, and the polymeric shell precisely tailored the drug release behavior in a pH-dependent manner. Similarly, an acid/oxidation dual-responsive polymer was designed and further applied in encapsulating atorvastatin-loaded PSi nanoparticles. The meticulously designed system not only obtained a dynamic drug release behavior, but also showed an orchestrated cascade that facilitated bio-mimetic diabetic wound healing. To better elucidate the biocompatibility of PSi for DDS fabrication, the biological effects and immunogenicity of different PSi nanoparticles were evaluated at pre-existing lesion sites, which provided insights for further applications of PSi in DDS fabrication. In conclusion, multiple PSi-based nanohybrids with different on-demand responses were fabricated and applied as DDSs for different diseases. The newly developed nanosystems tailored drug release and obtained multiple modalities, ranging from real-time bio-imaging to bio-mimetic/bio-response alteration, as such, represent promising platforms for future therapy regimes.
  • Fontana, Flavia (Helsingin yliopisto, 2019)
    Fontana F., 2019. Biohybrid Cloaked Nanovaccines for Immunotherapy Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis, 47/2019, pp.78 ISBN 987-951-51-5286-2 (Paperback), ISBN 978-951-51-5287-9 (PDF, http://ethesis.helsinki.fi), ISSN 2342-3161 Immunotherapy is revolutionizing cancer treatment achieving durable and long-term responses in patients. However, only subsets of patients treated experience a positive outcome, due to immunotherapeutic resistance. Combinations of immunotherapeutics can overcome the drug resistance; the administration of a cancer vaccine or an oncolytic virus followed by immune checkpoint inhibitors is under investigation. Thereby, there is an unmet need for powerful, yet safe vaccines. Nanoparticles, in particular porous silicon nanoparticles, present ideal characteristics to formulate nanovaccines, thanks to their size-specific targeting to the lymphoid organs, to their intrinsic adjuvant effect, and to the possibility to simultaneously load adjuvants and antigens. Moreover, biohybrid cell membrane technology has been proposed as an innovative antigenic source. Thus, the aims of the current thesis were to develop a biohybrid multistage nanovaccine formulation and to evaluate its anticancer efficacy in murine tumor models. Firstly, the parameters affecting the formulation of the biohybrid nanosystems were assessed, along with the elucidation of the influence of the cell membrane coating on the colloidal stability in physiological conditions and on the biocompatibility in different cell types. Secondly, the effect of the cell membrane-wrapping on the cellular uptake was evaluated in the presence of inhibitors of selective uptake pathways, to assess the differences between naked and coated nanoparticles. Then, a multistage nanovaccine was engineered by glass capillary microfluidics, followed by the cloaking with the cell membrane. The immunological profile of the nanovaccine was investigated in vitro, assessing the expression of co-stimulatory signals and the secretion of proinflammatory cytokines. The efficacy of the biohybrid nanovaccine as a monotherapy and in combination with an immune checkpoint inhibitor was then evaluated in melanoma murine models. Finally, the adjuvant core was changed from synthetic nanoparticles to oncolytic adenoviruses to investigate the translatability of the technique, the influence of the cell membrane-coating on the viral infectivity, and the preventive and therapeutic efficacy of the vaccine in different tumor models. Overall, porous silicon and adenovirus-based biohybrid nanovaccines were developed, providing new insights on the structure and efficacy of these systems as therapeutic cancer nanovaccines.
  • Julku, Ulrika (Helsingin yliopisto, 2019)
    Dopamine is one of the main neurotransmitters in the brain. Dopaminergic signalling regulates reward, memory, attention and motor functions. In the synapses of dopaminergic neurons, dopamine transporter (DAT) re-uptakes dopamine into the presynaptic nerve terminals after dopamine release terminating the dopaminergic signal and acting as one of the main regulators for kinetics of dopaminergic neurotransmission. Loss of dopaminergic neurons in the nigrostriatal pathway and protein aggregates called Lewy bodies are the main pathological findings in Parkinson´s disease. Lewy bodies are mainly composed of a protein called α-synuclein. The physiological role of α-synuclein has remained unclear but it has been suggested that the main function is regulation of dopaminergic neurotransmission since α-synuclein has been shown to participate in the regulation of dopamine synthesis, storage, release, and metabolism. α-synuclein-regulated functions in dopaminergic signaling are described in the literature review of this thesis. Prolyl oligopeptidase (PREP) is a serine protease that binds to α-synuclein and induces its aggregation. PREP inhibitors have beneficial effects in cellular and in vivo models of Parkinson´s disease by reducing α-synuclein aggregates and oligomers, and improving motor functions. Additionally, PREP inhibitors alter striatal dopamine level in mice and rats, and decrease immunoreactive DAT in the mouse striatum suggesting that PREP could have an effect on dopaminergic function. The aim of this study was to characterize the role of PREP in dopaminergic signaling and the effect of α-synuclein in PREP-mediated changes of the dopaminergic system. In the first study, the effect of PREP and α-synuclein on DAT phosphorylation and function was studied in DAT transfected HEK-293 cells. PREP altered DAT function and dopamine uptake, but the changes were not dependent on ERK phosphorylation or PKC activity. α-synclein had an effect on DAT phosphorylation in the absence of PREP but this was also independent of phosphorylation of ERK indicating that both α-synuclein and PREP are able to modulate DAT function via an ERK¬-independent mechanism. In the second study, the role of PREP in dopaminergic signaling was characterized in the nigrostriatal pathway of mouse. The influence of PREP was investigated by comparing the dopaminergic function of PREP knock-out mice and wild-type littermates. Lack of PREP elevated extracellular dopamine concentration, delayed re-uptake of dopamine, and increased phosphorylation of DAT in the mouse striatum indicating that PREP is able to regulate DAT function by modulating phosphorylation and localization of DAT. The effect of PREP inhibition on dopaminergic function, behavior, and α-synuclein in a Parkinson´s disease mouse model was investigated in the third study. Overexpression of α-synuclein was induced by supranigral microinjection of AAV-α-synuclein and mice were treated with the PREP inhibitor KYP-2047 after the onset of the behavioral symptoms. KYP-2047 treatment did not restore α-synuclein-induced reduction in striatal dopamine but behavioral improvement and reduction in α-synuclein oligomers indicated restoration of dopamine release and recycling. The aim of the fourth study was to investigate if α-synuclein-induced toxicity in the nigrostriatal pathway is dependent on PREP expression. The main finding was that α-synuclein toxicity was reduced in the absence of PREP and restoration of PREP expression increased toxicity in the behavioral tests. However, nigrostriatal dopamine level was not affected suggesting that lack of PREP protects dopamine release and recycling from α-synuclein-induced toxicity. In conclusion, PREP regulates DAT function in cells and in the mouse nigrostriatal pathway, but the mechanism is not dependent on ERK and PKC activation. Deletion of PREP or PREP inhibition do not have effects on α-synuclein-induced dopaminergic cell loss, but they are able to restore behavior and dopaminergic function in the mouse brain suggesting that PREP inhibitors could provide a novel treatment for Parkinson´s disease.
  • Albert, Katrina (Helsingin yliopisto, 2019)
    The neurodegenerative disorder Parkinson’s disease is diagnosed when motor symptoms appear, which is caused by death of the substantia nigra dopamine neurons. Most disease cases are idiopathic, and there are currently no disease-modifying therapies. Since the mechanism underlying Parkinson’s disease is still unknown, bringing treatments to the clinic has been difficult. Alpha-synuclein (α-syn) is a protein found abundantly in the central nervous system of vertebrates. Its importance for Parkinson’s disease was confirmed when it was discovered that mutations in the gene led to an autosomal dominant disease form and that it is the majority protein in what is considered a pathological marker of the disease, Lewy bodies. Cerebral dopamine neurotrophic factor (CDNF) is a conserved protein with neurotrophic-like properties. It has been shown to protect dopamine neurons in toxin models of Parkinson’s disease and is currently in Phase I/II clinical trials. It has not been tested in α-syn animal models and therefore the aim was to model α-syn-based Parkinson’s disease and to test whether CDNF can intervene with α-syn aggregation and has a therapeutic effect. We generated two models that used α-syn to model sporadic Parkinson’s disease and test CDNF on: adeno-associated virus (AAV) and preformed fibrils. We used an AAV to overexpress human wild-type α-syn and were able to model nigrostriatal dopamine loss accompanied by behavioural deficits. However, the variation in the success of the model was too high to consider it feasible to test CDNF on. This, combined with concerns about controls, led us to conclude that it may not be an ideal model of sporadic Parkinson’s disease. Using a preformed α-syn fibrils model to seed endogenous α-syn, we observed modest behavioural deficits that were ameliorated by CDNF, however the model did not result in dopamine neuron loss with the measures used. Although, we were able to model the spreading of Lewy body- and neurite-like inclusions that were positive for phosphorylated α-syn. From parallel in vitro studies we can conclude that CDNF is affecting the preformed α-syn fibrils model, but further studies are needed to clarify this. Since CDNF has been successful in the 6-hydroxydopamine (6-OHDA) model after striatal injection, we tested injection to the substantia nigra and characterized the injection in naïve rats to further study CDNF. We expected similar effects of CDNF on dopamine neurons and behaviour using nigral injection, however issues with the injection paradigm and that CDNF was given as a single injection meant only minor behavioural effects and no restoration of dopamine neurons. Though when CDNF was injected to the substantia nigra of naïve rats, it was not transported to the striatum, but rather diffused around the midbrain. Lastly, we used a proteasomal inhibitor, the lactacystin toxin. When lactacystin was injected we observed a buildup of α-syn, nigrostriatal dopamine loss, neuroinflammation, and mild behavioural deficits. In general, this was repeated successfully and could be used for therapeutic studies. In conclusion, we used four different methods to model Parkinson’s disease to varying degrees of success in order to test CDNF. Our results indicate the importance of having proper controls and outcome measures. Additionally, we had success in modeling the progressive spreading of Lewy-like pathology, a phenomenon that is occurring in Parkinson’s disease. Notably, CDNF had some success and future studies will explore this further.

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