Farmasian tiedekunta

 

Nyligen publicerat

  • Almeida, Patrick (Helsingin yliopisto, 2018)
    Recent breakthroughs in nanotechnology have paved the way for a new era in cancer medicine. Among the myriad of nanotechnology-based systems that have been revolutionizing the field of cancer nanomedicine, porous silicon (PSi) nanoparticles have recently emerged as a promising nanoplatform, owing to advantageous physicochemical and biological properties. Nevertheless, the successful establishment of PSi nanoparticulate systems as effective cancer nanomedicines is challenged by several shortcomings associated with the instability in biological fluids, the poor tumour targeting efficiency and unfavourable pharmacokinetics, the limited capacity to overcome extra and intracellular biological barriers, and the ubiquitous and uncontrolled release of the therapeutic payloads. This dissertation aimed at designing and developing novel strategies, including the surface modification of PSi nanoparticles with biofunctional polymers and the engineering of advanced multifunctional PSi-based nanocomposites, in order to overcome some of the aforementioned deadlocks, improving the tumour targeting and drug delivery efficiencies, and ultimately potentiating the application of PSi nanomaterials in cancer nanomedicine. First, the biofunctionalization of PSi nanoparticles with a hyaluronic acid (HA) derivative was proven to improve the colloidal and plasma stabilities and to significantly enhance the cellular internalization of the nanosystems in breast cancer cells. The HA-modified PSi nanoplatforms exhibited higher affinity and endocytic activity in the cells overexpressing the CD44 receptor, thus evidencing a great potential for further development as active targeted drug delivery systems to CD44-overexpressing tumours. Next, a bilayered zwitterionic PSi nanocomposite was fabricated by successive conjugation of polyethyleneimine and poly(methyl vinyl ether-alt-maleic acid) polymers on the surface of PSi nanoparticles. In addition to satisfactory cytocompatibility, and high colloidal and plasma stabilities, the designed polymeric surface modification was shown to enhance the non-specific cellular association and uptake, and to improve the intracellular trafficking of the PSi nanoparticles in breast cancer cells. Moreover, this strategy contributed to increase the drug loading of methotrexate (MTX), sustain the release of the drug and potentiate the in vitro antiproliferative effect of the MTX-loaded PSi nanocarriers. In addition, PSi nanoplatforms were used to engineer multifunctional PSi-based nanocomposites, envisioned for cancer therapeutic and theranostic applications. In one approach, both sorafenib-loaded PSi and gold nanoparticles were simultaneously encapsulated into a self-assembling polymeric nanocomplex. In another study, the same nanocomplexes were used to encapsulate DNA-capped PSi nanoparticles, as an innovative strategy to bioresponsively deliver hydrophilic and hydrophobic drug molecules into the cytosolic compartment of cancer cells. The potential of the fabricated multifunctional PSi-based nanocomposites stemed from the versatility to incorporate a combination of nanosystems, hydrophilic or hydrophobic drug molecules, and fluorescent dyes within a single nanostructure, and the capability to enhance the cellular interactions, endocytosis and cytoplasmic delivery of the encapsulated nanoparticles and therapeutics. In conclusion, the developed PSi-based nanocomposites exhibited great potential for cancer targeting and drug delivery, representing an advanced contribution for the successful implementation of PSi nanomaterials as the next generation of cancer nanomedicines.
  • Saarinen, Jukka (Helsingin yliopisto, 2018)
    In the pharmaceutical industry, novel analytical techniques are required to gain important insights about new drug candidates and their formulations as early as possible. This information can be used to develop more efficient, safe and also economically profitable medicines. Microscopy techniques can be used for example to follow the fate of nanoparticles in cells and tissues, but also to monitor the changes in solid-state forms of drug molecules during drug development and storage. The overall aim of the Thesis was to evaluate the capability of non-linear optical imaging, especially coherent anti-Stokes Raman scattering (CARS), second harmonic generation and sum-frequency generation (SHG and SFG) microscopies, in pharmaceutical applications including imaging of live cells, nanoparticle cellular uptake and pharmaceutical solid-state analysis. First, the capability of CARS microscopy to image live cell cultures on pharmaceutically relevant membrane inserts was evaluated. It was found that, label-free CARS microscopy can be used to image Caco-2 cells (used in drug permeation studies) grown on PTFE inserts in a non-destructive manner. Label-free CARS microscopy could probe lipid droplets and the size of lipid droplets increased substantially over a 21- day culturing period, which is important in the context of drug permeation studies, since lipid content of the cells will influence drug permeation. Next, CARS microscopy was successfully used to probe non-fluorescent nanocrystals in cells in a label-free and chemically-specific manner. In addition, electron microscopy could be used to visualize the subcellular location of nanocrystals in cells with nanometer spatial resolution in a chemically-specific manner by using a developed correlative imaging method with CARS and EM. At the end, multimodal CARS and SHG/SFG imaging was used to visualize the distribution and crystallization of indomethacin and its solid states on tablet surfaces. The combined use of two imaging modalities is beneficial, since data provided by using two techniques relying on different mechanisms (detection of molecular vibrations (CARS) and SFG signal produced by non-centrosymmetric crystals), can support each other. In summary, it was demonstrated that non-linear optical imaging can be a very useful tool in pharmaceutical applications including imaging of live cells, nanoparticle cellular uptake and solid-state analysis. The results were obtained by using a commercially available microscope, which suggests that there is plenty of potential in these techniques to be applied on a wider scale. The use of these techniques is likely to increase with further instrument commercialization in the near future.
  • Ferreira, Mónica (Helsingin yliopisto, 2017)
    Currently, there is no major discovery of an effective cure to restore the function of an injured heart, despite the existing and developing therapies. While existing options ameliorate the care of myocardial infarction (MI) and heart failure patients, cardiac stem cell therapy has only recently shown positive results in clinical trials, and thus there is an urgent medical need to develop advanced therapeutic entities to reverse this disease burden. The employment of biomaterials as potential therapeutics for MI is at the pre-clinical stage. Particulate systems are arising as a promising tool to provide minimally invasive treatment, an important aspect to take into account for clinical translation and patient compliance. Porous silicon (PSi) and spermine-acetalated dextran (AcDXSp) are emerging biomaterials for applications in varying biomedical fields. Drug delivery is one of these fields benefiting from the materials’ properties, such as biocompatibility, biodegradability, customized particle preparation, surface functionalization, simple yet efficient drug loading, and tunable release of the therapeutic cargos. Therefore, the aim of this thesis was to develop multifunctional PSi and AcDXSp platforms for targeted drug delivery to and imaging of the ischemic heart. Initially, the biocompatibility of PSi-based carriers of different sizes and surface chemistries was evaluated. Secondly, three different PSi-based nanosystems were developed, functionalized with a metal chelator for radiolabeling and three different peptides (atrial natriuretic peptide (ANP) and two other heart-homing peptides), with the aim to screen the targetability of the nanoparticles to the ischemic heart. All the nanosystems showed no toxicity up to 50 µg/mL concentration, and cell–nanoparticle interaction studies in cardiomyocytes and non-myocytes revealed a preferential cellular interaction with ANP-functionalized nanoparticles in both the cell types, through the natriuretic peptide receptors (NPRs) present at the cell surface. Thirdly, the ANP-PSi functionalized nanoparticles were PEGylated in order to improve the colloidal stability and enhance the circulation time. Upon labeling with radioisotope Indium-111, the ANP-PSi nanoparticles displayed a preferential accumulation and selectivity towards the endocardial layer of the ischemic heart. In vivo delivery of a cardioprotective small drug molecule from the ANP-PSi showed attenuation of the extracellular signal-regulated kinase pathway that is involved in the hypertrophic signaling of the injured heart. Lastly, and in parallel, the development of functionalized and dual-loaded AcDXSp nanoparticles for potential application in cellular reprogramming was proven successful, by utilizing acidic pH-triggered drug delivery of the two poorly water-soluble cargos. The incubation of non-myocytes with ANP-functionalized AcDXSp nanoparticles showed therapeutic modulation of key signaling pathways involved in the direct fibroblast reprogramming into cardiomyocytes. Overall, PSi and AcDXSp-based (nano)particulate systems were developed, bringing new insights about potential therapeutic advances in the applicability of imaging and targeted delivery of relevant pharmacological molecules to the ischemic heart with a minimally invasive therapeutic approach.
  • Räsänen, Riikka-Marjaana (Unigrafia, 2017)
    This thesis describes the applicability of different types of IMS instruments in the direct measurements of gaseous and solid samples and in fundamental studies of gas-phase ion chemistry. A handheld chemical detector containing an aspiration ion mobility spectrometry (AIMS) was applied in the monitoring of gas phase explosive triacetone triperoxide (TATP) from air flow. The instrument-normalized detection threshold (20 pA) was exceeded already with the lowest sample concentration of 0.3 mg m−3. The response time of the instrument was less than five seconds. AIMS was also used to monitor chemical changes in the headspaces of the chambers containing microbe contaminated and sterile particle board samples in humid conditions. It was possible to separate the distinct chemical profiles of the chambers with sterile and microbe-contaminated specimen by principal component analysis. Overall, AIMS was found to be an adequate technique in dynamic screening of TATP and in monitoring of the changes in the microbe metabolism. Ambient ionization techniques, direct analysis in real time (DART) and desorption atmospheric pressure photoionization (DAPPI), were combined with travelling wave ion mobility-mass spectrometry. In the surface analysis of almond, pharmaceuticals, vitamin tablets and dried blood spot sample, the ion mobility separation reduced the chemical noise in the mass spectra and increased the signal-to-noise ratio. In the comparative studies of DAPPI and DART ionization, the limits of detection were between 30−290 and 330−8200 fmol for DAPPI and DART, respectively, for the tested compounds bisphenol A, benzo[a]pyrene, ranitidine, cortisol, and α-tocopherol. Finally, the reactions of phenol and fluorinated phenols with Cl− in ambient pressure were investigated by drift tube ion mobility-mass spectrometry. For the least fluorinated phenols (phenol, 2-fluorophenol and 2,4-difluorophenol) with the lowest gas-phase acidities, the Cl− adducts [M+Cl]− and [2M+Cl]− were the major products in both low and high sample concentration. For the highly fluorinated phenols (2,3,6-trifluorophenol and pentafluorophenol), [M−H]− and [2M−H]− were the main products in high sample concentration. In low concentration [M−H]− and [M+Cl]− were the main products. In case of pentafluorophenol (PFP), in high temperature conditions the dimer was [2PFP−H]− instead of [2PFP+Cl]−. In conclusion, IMS has many advantages and application possibilities. It allows the rapid detection and continuous monitoring of volatiles directly from ambient air. IMS can also be used as a pre-separation technique in ambient mass spectrometry, without increasing the total analysis time remarkably. In IMS, it is also possible to study gas phase reactions in ambient conditions. Some of the IMS applications presented in this thesis could be developed further to be a permanent part of routine monitoring, analysis, and research work. For example, in the fundamental studies of phenols, the possibility to use updated versions of the instruments could improve the accuracy of the experiments. In addition, broader studies with several experimental conditions, would increase the possibility to develop the method further, especially in the monitoring of TATP, and building material and microbe emissions from the gas phase with AIMS.
  • Laine, Niina (Helsingin yliopisto, 2017)
    BACKGROUND AND OBJECTIVES Rational use of antimicrobials is paramount due to increasing bacterial resistance and a lack of novel antimicrobials. Investigating the clinical use and consumption of antimicrobials aids in the prudent use of these drugs in a tertiary paediatric hospital. The purpose of this study was to obtain detailed information on the use of antimicrobials in a tertiary Children’s Hospital, Helsinki University Hospital, in order to support prudent, safe and efficient use of antimicrobials. The objectives were the following: 1) To evaluate the appropriateness of antimicrobial therapy (AMT) in children with blood culture positive infections (Study I), 2) To investigate the consumption of antimicrobials in the hospital in Defined Daily Doses (Study II), 3) To record the prevalence of off-label use of antimicrobials in neonates (Study III) and last, 4) To analyse the occurrence of antimicrobial medication errors in children (Study IV). MATERIALS AND METHODS The Children’s Hospital, University of Helsinki, is a tertiary hospital in Finland. In Study I, data on 149 children (0–17 years) with blood culture positive hospital infections between 2005 and 2012 were collected. In Study II, the consumption of antimicrobials in Defined Daily Doses (DDDs according to the Anatomical Therapeutic Chemical (ATC)/DDD index) was investigated retrospectively between 2003 and 2013. In Study III, the prevalence of off-label use of antimicrobials was investigated in three different paediatric cohorts. The largest cohort consisted of premature NICU patients (450–2000g) with blood culture positive infections and antimicrobial therapy given between 2005 and 2014 (N=282). In Study IV, the types of documented antimicrobial errors were analysed. The errors were reported by healthcare professionals using a voluntary web-based error reporting system, HaiPro between 2009-2014. Overall, different types of methods were used regarding quantitative and qualitative analysis and retrospective reviews of electronic patient records and registry data. RESULTS The AMT was inappropriate in 17% (26/149) of patients with blood culture positive infections (Study I). Three of these patients received antimicrobials that were totally ineffective according to in vitro data. During 2003 and 2013, the use of many beta-lactam antimicrobials increased. The most notable change was in the use of carbapenems, which increased by 110% during the study period (Study II). A total of 18% (51/282) of premature neonates with blood culture positive infection received at least one off-label antimicrobial (Study III). An increase in birth weight was found to statistically significantly decrease the probability of off-label usage (odds ratio=0.85 for 100g increase in birth weight, p-value < 0.001). In Study IV, there were 157 antimicrobial errors reported in 149 patients from four wards (GEN, NICU, HEM-ONC and INF). Two errors were reported as clinically significant (2/149, 1%). Most of the errors occurred with drugs with high consumption, such as cefuroxime (15/157, 10%) and penicillin G (15/157, 10%). CONCLUSIONS These studies gave a useful overall picture regarding AMT and the use of antimicrobials at the Children’s Hospital. More attention should be paid to appropriate AMT, and training of prescribers should be provided. This thesis provides a window into issues that undermine the quality of care regarding hospital infections in paediatrics and aids the launch of an antimicrobial stewardship program (ASP) in the Children’s Hospital.
  • Tseng, Kuan-Yin (UNIVERSITY OF HELSINKI, 2017)
    Stroke is one of the leading causes of death and a major cause of disabilities in adults. More than half of stroke victims suffer some type of disability, ranging from different levels of minor weak- ness in a limb to a complete loss of mobility. Currently, treatment of stroke requires a stringent re- habilitation programs. Nevertheless, two thirds of all patients will still have some type of difficulty with regular daily activities. Recent experimental findings raise the possibility that functional improvement after stroke may be achieved through neuronal replacement by endogenous neural stem cells (NSCs) residing in the adult brain. Therefore, additional understanding of the properties of NSCs will help to identify their optimal potential in cell-based therapy. Neurotrophic factors are a family of proteins that are important in neuronal development and function, and have been studied as possible drugs for ischemic brain injury. In addition to Brain-Derived Neurotrophic Factor (BDNF) and Glial cell line-Derived Neurotrophic Factor (GDNF), Mescenphalic Astrocyte-Derived Neurotrophic Factor (MANF) and Cerebral Dopamine Neurotrophic Factor (CDNF), that form a distinct family of evolutionary conserved proteins with neuroprotective effects, have potential in the treatment of stroke. While MANF has been shown to protect cortical neurons from death in a rodent model of ischemic brain injury, the effects of post-stroke MANF ad- ministration on cellular processes during the recovery phase are poorly understood. To shed light on the possible regenerative potential of MANF for the injured brain, we need to first investigate the roles of endogenous MANF in neural stem cells (NSC) in a normal or pathological condition. We developed and optimized a work platform for studying the regulation and effect of MANF on biological properties of NSCs and cortical development. Our findings reveal an important role of MANF in neurite outgrowth and neuronal migration in the developing cortex. In addition, we demonstrated that endogenous MANF has the potential to protect NSCs against oxygen and glucose-deprivation conditions. Next, using neurosphere and subventricular zone (SVZ) explant cultures, we further studied the effect of MANF administration on cell differentiation and migration. We presented the data that exogenously added MANF can induce neural/glial differentiation and promote cell migration out of SVZ explants. Also, utilizing the advantage of NSCs as a target for MANF, we discovered that exogenous MANF can induce the phosphorylation of STAT3 in NSCs. Finally, we used the rat model of ischemic stroke to compare the effects of MANF and GDNF in neurogenesis after stroke. While injection of GDNF into lateral ventricle has a strong mitogenic effect to increase neurogenesis in SVZ, it does not induce migration of neuroblasts towards the ischemic area. In contrast, MANF facilitates the migration of neuroblasts towards the lesioned cortex. Regarding long-term infusions in the peri-infarct zone, both GDNF and MANF recruited the neuroblasts in the infarct area. However, only MANF accelerated functional recovery after stroke. In summary, this work has extended the knowledge of MANF’s capacity for neuronal differentiation as well as migration, and the regenerative capacity for its therapeutic use in further studies.
  • Lantto, Tiina A. (Helsingin yliopisto, 2017)
    Plant phenolics and extracts are able to affect cell signalling associated with regulated cell-death mechanisms. Such mechanisms play a crucial role in the normal homeostasis of an organism, but inadequately functioning cell-death machinery is a component of the development of complex diseases, such as cancer, where cells divide in an uncontrolled manner. Apoptosis is the most studied regulated cell-death mechanism associated with cancer. One of the key triggers of and contributors to apoptotic cell death is a tumour suppressor p53. This protein is constantly produced, though it is activated only by several cellular stress responses, such as endoplasmic reticulum stress. The purpose of this study is to investigate the cytotoxic and apoptotic properties of three plant phenolics – curcumin, resveratrol and quercetin – and seven plant extracts – basil, juniper, laurel, lemon balm, parsley, and Siberian pine – in cancerous neuroblastoma and melanoma, and non-cancerous fibroblast cell models. The emphasis of the work is on plant extracts due to their claimed additive or synergistic effects on cellular mechanisms. The effects of different treatments are determined by two cell-viability tests, followed by Western blot assays of the amounts of p53, anti-apoptotic Bcl-2, and inflammatory p65. Apoptotic events are defined by the activity of caspase 3 and DNA fragmentation. Further testing to reveal a broader spectrum of effects is defined by the cDNA RDA method in order to investigate genes expressed differently in treated and in untreated cells. The results of this study support existing knowledge of the effects of single-plant phenolics, and reveal new mechanisms for the activity of plant extracts. Possible synergistic or additive effects of juniper plant extract on apoptosis through endoplasmic reticulum stress are observed. Plant phenolics and extracts may provide a unique pool of drug candidates for the prevention or treatment of cancer. The use of plant extracts as drug candidates is especially interesting due to the possible synergistic or additive effects they achieve at low concentrations.
  • Pessi, Jenni (Helsingin yliopisto, 2017)
    This thesis consists of two parts, particle formation and analysis. In the first part, particle formation in microfluidic devices and in devices employing supercritical fluids is investigated, and in the second part, essential issues in analytical methods for determining drug release and solid-state properties are addressed. Microfluidic technology was employed to produce microcapsules for protein formulations. The microcapsules were produced with a biphasic flow to create water-oil-water double emulsion droplets with ultrathin shells. All the particles were found to be intact and with a particle size of 23 - 47 µm. The encapsulation efficiency of bovine serum albumin in the microcapsules was 84%. This study demonstrates that microfluidics is a powerful technique for engineering formulations for therapeutic proteins. A new, robust, stable, and reproducible method based on expansion of supercritical solutions using carbon dioxide as a solvent was developed to produce nanoparticles. The method, Controlled Expansion of Supercritical Solution (CESS), uses controlled mass transfer, flow, pressure reduction, and particle collection in dry ice. CESS offers control over the crystallization process as the pressure in the system is reduced according to a specific profile. Controlled pressure reduction keeps the particle growth and production process stable. With CESS, we produced piroxicam nanoparticles, 60 mg/h, featuring narrow size distribution (176 ± 53 nm). The Lyophilic Matrix (LM) method was developed for investigating dissolution rates of nanoparticles, powders, and particulate systems. The LM method is based on its ability to discriminate between non-dissolved particles and the dissolved species. In the LM method, the test substance is embedded in a thin lyophilic core-shell matrix. This permits rapid contact with the dissolution medium while inhibiting dispersion of non-dissolved particles without presenting a substantial diffusion barrier. By minimizing method-induced effects on the dissolution profile of nanopowders, the LM method overcomes shortcomings associated with current dissolution tests. Time-gated Raman spectroscopy was applied for solid-state analysis of fluorescent powder mixtures. A setup with a 128 × (2) × 4 CMOS SPAD detector was used for the quantitative analysis of solid-state forms of piroxicam. Time-gating provides an instrumental method for rejecting the fluorescence signal. This study demonstrated that traditional PLS analysis of time-gated Raman spectra resulted in mean RMSE of 4.1%. The time-gated Raman spectroscopy method shows potential for relatively routine quantitative solid-state analysis of photoluminescent pharmaceuticals.
  • Ruokolainen, Miina (Helsingin yliopisto, 2017)
    Redox reactions play an important role in human physiology and pathophysiology. For example, oxidative stress and free radical-mediated oxidation of proteins and lipids are implicated in several diseases such as Alzheimer’s and Parkinson’s disease. Oxidation reactions belong also to the most important phase I metabolism pathways of drugs, which can give rise to pharmacologically active or toxic metabolites. The established methods for in vitro drug metabolism studies, e.g. methods using hepatocytes, human liver microsomes (HLMs), and recombinant enzymes, are relatively time-consuming and expensive. Thus, the potential of several nonenzymatic oxidation methods, such as those based on metalloporphyrins, electrochemistry (EC), and Fenton reaction, have been explored for metabolism studies. However, new methods need to be developed to enable rapid production of drug metabolite standards and since none of the above nonenzymatic methods allow comprehensive prediction of phase I drug metabolism. The titanium dioxide (TiO2) photocatalysis method was developed and applied to evaluate the effect of phosphorylation of tyrosine on the oxidation of (phospho)peptides with the same sequence but different phosphorylation states. The results obtained using ultra-high-performance liquid chromatography – mass spectrometry (UHPLC-MS) show that nonphosphorylated tyrosine was the amino acid most susceptible to hydroxyl radical-initiated oxidation, but oxidation of tyrosine was in most cases inhibited by its phosphorylation. The feasibility of TiO2 photocatalysis for imitation of in vitro phase I HLM metabolism of small drug molecules was studied using UHPLC-MS and compared with the electrochemically assisted Fenton reaction (EC-Fenton) and EC. TiO2 photocatalysis, EC-Fenton, and EC imitated 44%, 31%, and 11%, respectively, of the in vitro phase I HLM metabolites of four model compounds. As TiO2 photocatalysis proved most feasible for the imitation of in vitro phase I HLM metabolism, its feasibility for imitation of in vitro phase I HLM metabolism of five anabolic steroids was also examined. TiO2 photocatalysis was able to imitate over half of the hydroxylation and dehydrogenation metabolites, but its imitation of the metabolites resulting from combinations of these reactions was considerably poorer. To enable even more rapid experiments to study biologically relevant oxidation reactions, TiO2-photocatalysis was simply integrated with desorption electrospray ionization (DESI)-MS by using the same TiO2-coated glass wafer for photocatalytic reactions and DESI-MS analysis. This new method enabled high-throughput investigation of photocatalytic oxidation reactions, as demonstrated using 12 model compounds, and imitation of several drug metabolism reactions of three model compounds studied in more detail. In conclusion, TiO2 photocatalysis proved a feasible method for oxidation of compounds with different polarities. TiO2 photocatalysis cannot predict drug metabolism comprehensively, but offers a potential method for rapid, simple, and inexpensive study of oxidation reactions of biomolecules and imitation of several drug metabolism reactions. Preparative scale synthesis of oxidation products by TiO2 photocatalysis is likely an alternative application of the method, but this remains to be demonstrated.
  • Penttinen, Anna-Maija (Helsingin yliopisto, 2017)
    Parkinson’s disease (PD) is a neurodegenerative disease characterized by intracellular proteinaceous inclusions called Lewy bodies and progressive loss of dopaminergic neurons in the substantia nigra (SN). The first symptoms of PD are non-motor, such as hyposmia and gastrointestinal disturbances, followed by motor symptoms, such as tremor and rigidity. Currently available therapies, medication, surgical procedures and supportive therapies, are symptomatic and do not affect the underlying cause of the disease — the neuronal degeneration. Thus, a new therapy which would restore the dopaminergic phenotype of dying neurons and thus slow down or even halt the progress of the disease is needed. Neurotrophic factors are secretory proteins regulating survival and functioning of the neurons as well as the formation of new neuronal contacts. Neurotrophic factors have shown great potential in animal models of PD, but in clinical trials, the results have been contradictory. One possible explanation for this is poor diffusion and bioavailability of the therapeutic proteins in the target tissue. The aim of this study was to explore the neuroprotective effects of the isoforms of two of the most potent dopamine neurotrophic factors, GDNF (glial cell line-derived neurotrophic factor) and its homolog neurturin (NRTN) in an experimental model of PD, and to characterize a new stable low-dose 6-hydroxydopamine (6-OHDA) rat PD model. In the PD model the degeneration of the nigrostriatal pathway was induced by administrating toxic dopamine analog 6-OHDA into the striatum, where the nerve terminals of the dopaminergic neurons are located. We compared several different administration paradigms to find the optimal parameters to induce a stable lesion model with high success rate. The cell loss induced with low doses (6-9 µg) of 6-OHDA was at similar level as the cell loss induced with higher (20 µg) doses of 6-OHDA. The advantage of using low 6-OHDA doses is the avoidance of non-specific damage, which occurs with higher 6-OHDA doses. Moreover, the low-dose induced lesions have high success rate, reducing the number of animals needed in the experiments and increasing the reliability of the obtained results. The spreading of NRTN in the brain tissue was improved by modifying the extracellular matrix binding sequence of the protein. New NRTN variants were biologically active and were able to initiate signaling via tyrosine kinase Ret (rearranged during transfection). In the neuroprotection assay in rat 6-OHDA model of PD NRTN variant N4 protected the dopaminergic neurons in the SN and fibers in the striatum as well as improved the motor behavior of the animals. In neurorestoration assay, N4 showed a trend in improving the behavioral deficits of the animals. GDNF, on the other hand, was administered to the brain with viral vectors, enabling long-term protein expression in the target tissue. GDNF has been widely studied, but the research has focused on the full-length constitutively secreted α-isoform, whereas the biology of the shorter and activity-dependently secreted β-GDNF has not been studied in vivo before. In the non-lesioned striatum, both isoforms increased striatal dopamine transporter-immunoreactivity. Both isoforms also protected the dopaminergic neurons in SN from 6-OHDA-induced degeneration. The results show that these new and less studied neurotrophic factor isoforms are able to slow down the degeneration of the midbrain dopaminergic neurons. In other words, both NRTN variant N4 and β-GDNF are potential disease-modifying factors for PD.
  • Suvikas-Peltonen, Eeva (Helsingin yliopisto, 2017)
    In hospital care, many medicines require aseptic compounding or preparation before they are administered to patients. The parenteral administration of microbiologically contaminated doses can result in bacteriaemia, other morbidity and even death. The aim os this study was to review the literature for incorrect practices in aseptic drug preparation and administration and recommendations for safer practices in hospitals. The aim was also to develop and content validate an assessment tool for medicine compounding on hospital wards. And at the final stage of this study, the aim was to audit compounding of intravenous medicines on hospital wards by using the developed assessment tool and take microbiological tests for identifying issues posing patient safety risks. A systematic literature search was conducted in PubMed. The first draft of the audittool was based on ISMP “Guidelines for safe preparation of sterile compounds” and a systematic literature search. The tool was then validated by using a two-rounded Delphi-method. A hospital pharmacist conducted external audit based on observation and interviews by using this validated 65-item assessment tool on 20 wards of a secondary care hospital in Finland. In addition microbiological test were collected. Associations between microbial sample results and audit tool results were discussed. In the review, the most reported incorrect practices were multiple use of vials and syringes and lack of overall disinfection during the aseptic preparation and administration. Recommendations to avoid contamination were classified into categories: equipment and medicines; disinfection; working envi-ronment; storing; catheter care; and quality of prepared medicines. The final audittool comprises of 64 items under the following topics: General principles of good compounding practices, Recording and con-firming medicine orders on the wards, Storage of medicines on the wards, Aseptic compounding of intravenous medicines and Quality assurance. The best practices related to logistic practices and quality assurance. Most of the obviated practices related to aseptic practices. All media fill tests were clean but in some of glove samples and settle plate samples were microbial growth. More contamination was found in wards where environmental conditions were inadequate or the use of gloves was incorrect.
  • Regina, Sirpa (Helsingin yliopisto, 2017)
    The objective of this doctoral dissertation is to present views, experiences and practices mainly related to the working-age insomniac, insomnia, and the treatment of it, as expressed by pharmacists working in Finnish community pharmacies. The specific aim is to investigate whether pharmacists see the pharmacy as having an important role in the initial stages of treating those who experience insomnia and are seeking to cure it. With regard to the self-treatment of insomnia, the study examines the kind of non-pharmacological advice offered by pharmacists when recommending self-care products and whether or not any underlying causes are being discussed with the customer. A further aim is to investigate what kind of pharmaceutical advice pharmacists provide on hypnotics and the kind of practices they employ in dispensing medicine to individual customers. Pharmacists’ views on working-age people who use hypnotics are also explored. The study was conducted as a qualitative cross-sectional study. The participants were pharmacists working at community pharmacies in mainland Finland (n = 188–277). The data was collected from five online surveys carried out in the spring of 2013. The participants were contacted using the mailing lists of the Finnish Pharmacists’ Association’s member organisations. The surveys could be accessed via a link sent by e-mail. The key questions were open-ended so as to obtain in-depth information on the pharmacists’ views, experiences and practices related to the research topic. The data was analysed mainly by using thematic analysis and both deductive and inductive content analysis. SPSS statistical software was also utilised in the analysis process. According to the study, pharmacists did not consider the pharmacy to be an obvious first choice for providing help to those with insomnia. Determining the cause of insomnia was not considered by all pharmacists to be part of their remit, nor did they necessarily even think it was their job to find out why the customer used hypnotics. Pharmacists did not always automatically provide non-pharmacological advice on insomnia in connection with self-care products, and the treatment instructions they provided differed in both quantity and quality. Even for customers buying hypnotics for the first time, the advice related to the possible side effect, addiction, varied greatly from one pharmacist to the another. Customers whose use of hypnotics exceeded the recommended dose also received very different levels of support when collecting their next batch of hypnotics, since the pharmacists had different ways of dealing with this kind of situation. Pharmacists criticised medical doctors for their treatment practices regarding insomnia. The issue generating the most criticism was deviation from the clinical guidelines for treating insomnia. According to this doctoral dissertation, those with insomnia are far from receiving equal treatment when seeking help at a pharmacy. The quality of service received by customers with sleep problems depends on the pharmacist serving them, on the pharmacist’s competence, and on their motivation to collaborate with the customer. On the other hand, the pharmacists’ positive attitude towards non-pharmacological self-care for insomnia creates opportunities for expanding the range of services provided by pharmacies. More and more frequently, the care pathway for those experiencing insomnia could thus start at the pharmacy. This does, however, require that pharmacists receive further training. Some pharmacists also need to obtain a basic knowledge of insomnia treatment as well as information on doctors’ treatment practices for insomnia.
  • Sjöstedt, Noora (Helsingin yliopisto, 2017)
    Transport proteins embedded in the cell membranes of many organs can affect the absorption, distribution and elimination of numerous drugs. This can lead to the enhanced or restricted uptake or distribution of the drugs, nonlinear pharmacokinetics, transporter-mediated drug-drug interactions (DDIs) and inter-individual variability. Transporters may therefore alter the safety and efficacy of drugs, thus it is important to study drug-transporter interactions in drug development. The breast cancer resistance protein (BCRP, ABCG2) is one of the transporters involved in drug disposition. It belongs to the ATP-binding cassette (ABC) transporter family and uses ATP to expel drugs and other substrates out from cells. BCRP was initially found to cause drug resistance in cancer cell lines, but it is also expressed in healthy tissues such as the intestine, liver and blood-brain barrier, where it is one of the transporters limiting the uptake of many structurally diverse compounds. Despite interest in BCRP and other ABC transporters, it remains poorly understood how they recognize their substrates and which chemical structures are liable to interaction. In this thesis, a vesicle-based in vitro method was used to study the ligand preferences of BCRP. The results were compared to those obtained for the multidrug resistance associated protein 2 (MRP2, ABCC2), which is also implicated in drug transport. The results show that a range of natural compounds and their derivatives are able to inhibit BCRP transport and among these, flavonoids were identified as the most important group for inhibition. Conversely, MRP2 transport was affected by only few of the tested compounds. However, a more similar pattern of inhibition was seen for the two transporters when selected food additives were studied, where several food colourants were identified as inhibitors. In addition, the effect of one assay component (bovine serum albumin, BSA) on the in vitro transport kinetics of BCRP and MRP2 was evaluated. The inclusion of BSA in the vesicle assay lead to moderate changes (up to 2-fold) in transport activity, but the effects on in vitro − in vivo extrapolation are expected to be minor, at least based on the tested compounds. Finally, the vesicle assay was used to study the functionality of selected BCRP variants with polymorphisms in the transmembrane helices and they were found to have significantly impaired transport activity and expression compared to wild type BCRP. In summary, the vesicle-based transport assay was successfully applied to identify and evaluate the effects that BCRP interactions may have on the pharmacokinetics of BCRP substrates.
  • Talka, Reeta (Helsingin yliopisto, 2017)
    Tobacco use is the leading cause of preventable death worldwide. Nicotine is the primary addictive component of tobacco, and repeated nicotine exposure often leads to dependence in humans. Nicotine is one of the most commonly co-used substances among polysubstance abuse patients and combined use of nicotine and other drugs of abuse, such as opioids, increases the use of one or both substances. The health consequences associated with polysubstance abuse exceed those of either drug alone. The current pharmacotherapeutic options are ineffective among opioid-substituted patients and the levels of successful smoking cessation are low. At the cellular level, nicotine and opioids have their own molecular mechanisms of action, yet both drugs increase the activity of the reward pathway by increasing dopamine transmission. The purpose of these studies was to investigate the possible effects of different opioid ligands on human neuronal nicotinic acetylcholine receptors (nAChRs) expressed in cell cultures. Our results showed that morphine has a partial agonist effect at α4β2 nAChRs, a very weak antagonist effect at α3* nAChRs and a positive synergistic effect with nicotine on α7 nAChR function. We found that methadone acts as a non-competitive antagonist (NCA) at α4β2 and α3* nAChRs. We also confirmed that methadone is a human α7 nAChR agonist. In the prolonged studies with methadone and morphine, we found that human α3*, α4β2 and α7 nAChRs are differentially regulated by prolonged exposure to methadone and morphine. Buprenorphine was shown to be a weak antagonist at α4β2, α3*, and α7 nAChRs, and codeine had a positive modulatory effect on α4β2 nAChRs and a weak NCA effect on α3* nAChRs. Oxycodone seemed to have a mixed competitive/non-competitive effect on α4β2 nAChRs and a weak NCA effect on α3* nAChRs. Tramadol was shown to be a NCA of α3* nAChRs and a weak NCA of α4β2 nAChRs. Naloxone and naltrexone were mixed competitive/non-competitive antagonists of α4β2 nAChRs, weak NCAs of α3* nAChRs and weak antagonists of α7 nAChRs. Taken together, these studies showed that many opioid ligands have effects on nAChRs that are independent of their agonist or antagonist properties at opioid receptors. These findings suggest that some effects of the nicotine opioid interaction seen in humans can be partially mediated through the receptor-level interplay of these substances. These results, together with earlier findings, highlight the complexity of different nAChRs and the multiplicity of responses to opioid ligands.
  • Helfenstein, Andreas (Helsingin yliopisto, 2017)
    Som ett svar på bristen av verksamma antibiotika, håller läkemedelsforskningen ständigt på och letar efter nya idéer för att bekämpa mikroorganismer. Emellertid har växter, svampar och bakterier utvecklat en stor repertoar av överlevnadsstrategier för att gardera sig mot mikroorganismer. Dessa strategier kan stå som grund för utvecklingen av nya antibiotika. Ett första steg i antibiotikaforskning kan därför vara så kallad screening, där man undersöker möjlig antibiotisk verksamhet hos många olika kemiska substanser. I första delen av avhandlingen beskrivs hur vi använde screening för att undersöka substanser från olika träd. Data från det ovannämnda experimentet organiserades med ett software-verktyg som utvecklades speciellt för detta ändamål. Efteråt användes maskininlärning (en sorts artificiell intelligens) för att leta efter dold information i undersökningsresultaten. I avhandlingens sista del beskrivs hur information från offentliga källor, i det här fallet från bloggar, kan hjälpa och stöda farmaceutisk forskning. Vi utvecklade och använde ett verktyg som analyserar hur ofta olika växter nämns i samband med olika sjukdomar i blogginlägg. I samband med screeningen hittades två substanser som är verksamma mot Staphylococcus aureus, en bakterie som orsakar många infektioner i både hem- och sjukhusförhållanden. Insikterna från arbetet med projekten i maskininlärning och bloggtextanalys kan fungera som stöd för framtida projekt i antibiotikaforskning.