Farmasian tiedekunta


Recent Submissions

  • Helfenstein, Andreas (Helsingin yliopisto, 2017)
    Responding to the aggravating shortage of potent antibiotics, pharmaceutical research is constantly looking for new ideas to combat and contain perilous micro-organisms. As a result of persistent selective pressure, evolution has bestowed a powerful arsenal of antimicrobial survival strategies upon plants, bacteria and fungi. Screening of natural product libraries is an obvious and promising first step in antimicrobial research. We followed that approach by investigating the antimicrobial bioactivity of a set of abietane derivatives. To accommodate for the data that resulted from that screening, we then developed a tool to automate and integrate the data analysis and presentation into the screening workflow. In an attempt to further streamline the process, we evaluated identification techniques for micro-organisms growing in co-cultures based on machine learning. Including the right material in the screening is a key element for a successful campaign. In our last study, we analyzed user-generated online sources to search for potential leads for new anti-infectious drugs. The screening resulted in a potential new compound active against Staphylococcus aureus with moderate cytotoxicity. The abietane derivative showed a minimal inhibitory concentration of 60 µg/ml against S. aureus and 8 µg/ml against multi-resistant S. aureus. Machine learning is used more and more in natural product research, and our study suggested that random forest classifiers and support vector machines are efficient tools to identify micro-organisms from polymicrobial cultures. The proposed models had prediction rates > 90 % in the determination of growth for microbes in polymicrobial cultures. The Internet is an immense and ever-growing resource of information, and we sketched a possible way for how this data can complement the search of antimicrobial plant preparations. In three examples, we showed correlations between mentions of plants and medical terms. Centered on antimicrobial screening, this thesis studies different strategies to enhance the screening process through the help of bioinformatics. We developed, tested and used different tools and methods to support and facilitate library design, detection techniques, and data management. These are only small pieces in the search of new antimicrobial compounds, but they show that there are still many possible approaches to follow.
  • Holmström, Anna-Riia (Helsingin yliopisto, 2017)
    Medication errors are one of the most common incidents leading to adverse events in healthcare worldwide. Tackling these major problems requires the implementation of a systems approach to healthcare, stating that risks should be managed proactively by improving the healthcare system. One of the recommended key strategies for learning from medication errors and risk prone processes is the establishment of local and national medication error reporting (MER) systems in healthcare. This study explored national and local MER systems in different countries and what makes them work in learning from medication errors. The study also explored how continuing education in medication safety could be organised for practicing healthcare professionals. The study applied both qualitative and quantitative research methods and utilized various data sources. The study was based on the theory of Human Error and the systems approach to risk management. The study comprised of three phases. Phase I explored the existing MER systems in different countries and their development and implementation. 16 medication safety experts from different countries responded to an online-survey. A national or local MER system existed in 11 of the countries. Blaming for errors, and a lack of time, training and coordination of reporting continue to be the major barriers to reporting. Learning from errors and a non-punitive approach are essential features of a MER system. There is also a need for promoting international networking of medication safety experts and bodies for sharing information and learning from others. Several factors associated with the successful development and implementation of MER systems were also identified. Phase II assessed the inter-rater reliability of medication error classifications in a voluntary Reporting System for Safety Incidents in Health Care Organizations (HaiPro) widely used in Finland. Also medication errors (n=32 592) reported in 2007-2009 and their contributing factors were explored. The inter-rater reliability was found acceptable (κ ≥0.41) in 11 out of 42 (26%) variables (e.g., near miss or actual error) describing the reported medication errors. Thus, the medication errors reaching the acceptable level of inter-rater reliability could be pooled from different healthcare units for the exploration of medication errors at the level of all reporting organisations. The most frequently reported medication errors were: dispensing errors (33%, n=10 906); administration errors (24%, n=7 972); and documentation errors (17%, n=5 641). The most commonly reported contributing factor was deficiencies in communication and course of information related to patients medications. In Phase III educational approaches were developed for introducing medication safety for healthcare professionals as a three-day interdisciplinary course. International higher education experts in pharmacy (n=19) brainstormed four syllabi with teaching and assessment methods. Following this, a combined syllabus was developed. All four syllabi were based on constructive, problem-based learning methods and focused on understanding a systems approach in managing medication safety. Learning linked to learners practice through assignments at the workplace appeared to be the key. The study suggests that MER systems need to be improved in many countries together with their operational environments. Moreover, the operational environments of MER systems must support the functionality of these systems. The key factor for successful MER systems and learning from medication errors is having a systems approach as a theoretical context in all reporting and learning processes throughout the operational environments of MER systems. The current work also suggests that constructive problem-based learning linked to learners practice through assignments is the key when developing a course for continuing education in medication safety for healthcare professionals.
  • Hautala, Jaana (Helsingin yliopisto, 2017)
    Product acceptability and administration of drugs on a free choice basis are key factors determining the success of peroral veterinary medication, and are particularly important when treating chronic diseases of companion animals. Veterinary medicines found unpalatable and repulsive in odour, taste or form will result in refused voluntary intake. Particularly with cats, the issues related to product unacceptability are difficult to overcome, and specific tailor-made peroral dosage forms for cats are currently lacking. Both animal treatment compliance and owner treatment bonding are essential for successful veterinary drug therapy. Product dosing should be simple and easy, and it should be performed without any complications. The present study was undertaken to investigate feline peroral drug therapy and to develop novel feline-specific minitablets with increased palatability supporting the safe, simple, flexible and convenient drug treatment of cats. The acceptability of minitablets was evaluated in a new feline behavioural test setting with domestic pet cats. The minitablets, developed with a focus on target species characteristics, were found to be more acceptable than non-favoured food. However, improvements in minitablet odour and/or taste were required. Feline-specific flavours of non-natural origin together with a model substance having a bitter taste were investigated in the minitablet formulations. For a pharmaceutical industry point of view, synthetic flavours are considered more suitable over natural substances. In the present study, amino acids such as L-leucine and L-methionine, and thiamine hydrochloride, were considered as suitable candidates for feline minitablet formulations. New flavoured polymer coating formulations for feline medication purposes on minitablets were developed. Feline-specific synthetic flavours and their mixtures were incorporated in the aqueous film coatings of the polymethacrylate copolymer of Eudragit® E. The film coatings containing meat flavours of 2-acetylpyridine and 2-acetylthiazole in small concentrations were found the most applicable for minitablet coating and taste-masking purposes. Atomic layer deposition (ALD) was investigated as a novel ultrathin-coating method for pharmaceutical minitablets and for taste masking applications. The ALD thin coating, however, did not provide effective taste masking (with the coating levels studied) for the bitter tasting minitablets composed of heterogeneous excipients. In conclusion, the present results support the more cost-effective product development of palatable feline peroral medication. It is evident that pet and owner compliance as well as drug treatment efficacy and safety can be increased with the feline-specific products introduced here. The present results are also likely to be applicable for other veterinary target species, such as pet dogs. However, minitablets as dosage forms would additionally be suitable for humans.
  • Lázaro Ibáñez, Elisa (2017)
    Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies are a heterogeneous population of membrane particles released by cells to the extracellular space and into biofluids during normal physiological and pathological processes. EVs have been recognized as powerful vehicles for intercellular communication due to their capacity to transfer lipids, proteins, and nucleic acids, thereby influencing the properties and functions of recipient cells. Cells generate EVs with a unique composition based on their characteristics, which has a special relevance in the study of diseases such as cancer. Since specific molecular signatures can be passed on to tumor EVs, they are prime candidates for implementation as cancer biomarkers and in the delivery of therapeutics. Thus, exhaustive research is currently targeted towards elucidating the role of EVs in cell-to-cell communication and their therapeutic and diagnostic use. This thesis aims at broadening our understanding of the applicability and functional relevance of the use of EVs as prostate cancer biomarkers and therapeutic delivery vehicles. First, the practical use of EVs as a source of nucleic acid biomarkers in prostate cancer was assessed by exploring the DNA and RNA content of vesicles. Genomic DNA analysis of apoptotic bodies, microvesicles, and exosomes were performed to detect mutations within the EV cargo. The results were validated in plasma EVs of prostate cancer patients, from which the presence of prostate cancer-relevant genes was identified. Next, the prostate cancer-specific messenger RNA signatures of microvesicles and exosomes were analyzed. Unique nucleic acid signatures distinctive for the cell origin were found in the form of differential levels of mRNA transcripts from EV subpopulations. Overall, the nucleic acid content of EVs provided a new source of diagnostic information that could contribute to early prediction and diagnosis of prostate cancer, especially if combined. The role of EV-mediated intercellular communication was shown by comparing the uptake efficiencies and functional effects of EVs from prostate cancer cells of different metastatic status with non-cancer EVs. Additionally, the ability of EVs to carry and deliver a chemotherapeutic drug, together with their cytotoxic effects on prostate cancer cells were also analyzed. While EV uptake, in general, was an active and continuous process, the internalization rate and the subsequent functional effects of EVs on recipient cells differed based on the vesicle origin. EVs derived from cells of a metastatic source were more efficiently internalized than primary prostate cancer or benign prostate epithelial cell-derived EVs. Similarly, those EVs also induced a more proliferative and migratory phenotype in the recipient cells. Applying prostate cancer EVs in the in vitro delivery of paclitaxel to prostate cancer cells, resulted in an enhanced cytotoxic effect of paclitaxel mediated by EV delivery compared to the free drug. In summary, the results presented in this thesis support the concept that EVs can be utilized in both biomarker discovery and drug delivery fields as multifunctional tools for diagnosis and treatment of diseases such as prostate cancer. The studies presented here will also contribute to set the bases for further functional analysis of the roles of EVs in cell-to-cell communication. This new era of research could lead to faster, non-invasive, and more individualized diagnosis and improved treatments tailored to the specific needs of the patients.
  • Subramanian, Vigneshwari (Helsingin yliopisto, 2016)
    Designing drugs that are selective is crucial in pharmaceutical research to avoid unwanted side effects. To decipher selectivity of drug targets, computational approaches that utilize the sequence and structural information of the protein binding pockets are frequently exploited. In addition to methods that rely only on protein information, quantitative approaches such as proteochemometrics (PCM) use the combination of protein and ligand descriptions to derive quantitative relationships with binding affinity. PCM aims to explain cross-interactions between the different proteins and ligands, hence facilitating our understanding of selectivity. The main goal of this dissertation is to develop and apply field-based PCM to improve the understanding of relevant molecular interactions through visual illustrations. Field-based description that depends on the 3D structural information of proteins enhances visual interpretability of PCM models relative to the frequently used sequence-based descriptors for proteins. In these field-based PCM studies, knowledge-based fields that explain polarity and lipophilicity of the binding pockets and WaterMap-derived fields that elucidate the positions and energetics of water molecules are used together with the various 2D / 3D ligand descriptors to investigate the selectivity profiles of kinases and serine proteases. Field-based PCM is first applied to protein kinases, for which designing selective inhibitors has always been a challenge, owing to their highly similar ATP binding pockets. Our studies show that the method could be successfully applied to pinpoint the regions influencing the binding affinity and selectivity of kinases. As an extension of the initial studies conducted on a set of 50 kinases and 80 inhibitors, field-based PCM was used to build classification models on a large dataset (95 kinases and 1572 inhibitors) to distinguish active from inactive ligands. The prediction of the bioactivities of external test set compounds or kinases with accuracies over 80% (Matthews correlation coefficient, MCC: ~0.50) and area under the ROC curve (AUC) above 0.8 together with the visual inspection of the regions promoting activity demonstrates the ability of field-based PCM to generate both predictive and visually interpretable models. Further, the application of this method to serine proteases provides an overview of the sub-pocket specificities, which is crucial for inhibitor design. Additionally, alignment-independent Zernike descriptors derived from fields were used in PCM models to study the influence of protein superimpositions on field comparisons and subsequent PCM modelling.
  • Rojalin, Tatu (Helsingin yliopisto, 2016)
    Biophotonics is an emerging area of scientific research that uses light of photons to probe biological specimens, such as tissues, cells and molecules. The field of biophotonics is broad and considerably multidisciplinary. Therefore the prerequisite for understanding biophotonics is the capability to integrate the fundamental knowledge of the physics of light with perspectives of engineering of devices and instruments used to generate, modify, and manipulate light. Also, the fundamentals of biology and medicine are essential particularly comprehension of the biochemical and cellular phenomena that occur in living systems, and how such phenomena can be scaled up to concern the physiology of organisms, for example humans. Biological pathways and processes differ in the healthy and diseased state, and that is why it is essential to develop understanding of pathophysiology and various states of disease such as cancer, neurodegenerative disease or infectious states. Consequently, solid insights into the functions of medical treatments, including biopharmaceutics, are needed. Raman and surface plasmon resonance (SPR) are both light-based technologies enabling label-free measurements with high sensitivity. The primary aim of this Thesis was to tackle the emerging hardships encountered in the fundamental biopharmaceutical research, clinical settings, or in the pharmaceutical industry by introducing applications and data analysis methods based on the cutting edge Raman and SPR technologies. These techniques represent the biophotonic cornerstones as tools for biopharmaceutical applications throughout this Thesis. The scope of this Thesis was essentially broad. First, small drug molecules were investigated with state-of-the-art time-gated Raman technology, showing that the interfering photoluminescent backgrounds can be effectively suppressed thus improving the acquired Raman data significantly. Additionally, EVs were studied with laser tweezers Raman spectroscopy (LTRS) as larger scale analytes and representatives of a highly interesting topic in the current nanomedical field. When Raman data from several different types of single EVs was examined using sophisticated data analysis, distinct subpopulations were observed, and the differences could be related to the biochemical compositions of the vesicle membranes. For the first time, the study showed the importance of measuring single EVs instead of a pool of vesicles. Multi-parametric SPR (MP-SPR) technology was harnessed to develop applications and data analysis methods for small and larger scale analytes. Hence, a new small drug molecule, spin-labeled fluorene (SLF), was investigated in the context of Alzheimer s disease (AD) particularly its potential to interfere with the detrimental amyloid peptide aggregation processes. The developed bio-functional in vitro platform in combination with rigorous data analysis and computational simulations demonstrated the capabilities of SLF when it was employed in various biomimetic aggregation schemes. Moreover, liposomes were examined with the MP-SPR as larger scale nanomedical particles for the purposes of safe and effective nanocarrier development. The administration of a liposomal nanocarrier into the blood circulation was mimicked in the designed bionanophotonic in vitro schemes. Undiluted serum was made to interact with immobilized model liposomes in dynamic flow conditions. The findings revealed that the variation in surface chemistries of the liposomes plays a role when serum essentially immune system components are interacting with the liposomes. In particular, distinct soft and hard protein coronas were observed and characterized during the interactions. Collectively, the results and findings in this Thesis underline the broad potential of biophotonics for biopharmaceutical applications. The technical improvements in instrumentation, and creativity in the application and data analysis development make the future of biophotonics bright.
  • Karhu, Elina (Helsingin yliopisto, 2016)
    Chlamydia pneumoniae (C. pneumoniae) and Chlamydia trachomatis (C. trachomatis) are among the most common infectious microbes in humans. C. pneumoniae represents approximately 10% of community-acquired pneumonias worldwide. Statistically, everyone becomes infected 2-3 times during their lifetimes. The persistent form of C. pneumoniae is associated with other chronic diseases, such as asthma and chronic obstructive pulmonary disease. C. trachomatis is a sexually transmitted pathogen, which may result in infertility and ectopic pregnancies. Of all the infective agents it is also the one to cause most cases of blindness. The chronic forms of Chlamydiae cannot be cured by antibiotics, and a vaccine against them does not exist. Acute forms can be treated with antibiotics; but, resistance is increasing, given that treatment of Chlamydiae relies on wide-spectrum antibiotics. In this thesis new biologically active extract and compounds from natural sources were found to inhibit the growth of C. pneumoniae and C. trachomatis, the antichlamydial activity against C. pneumoniae being higher with all of the studied compounds and an extract. The antichlamydial effect of six lignans from the berries of Schisandra chinensis (Turczaninov) Baillon and their possible role as lead -compounds in the early drug discovery process were investigated. An aqueous methanol extract from Schisandra chinensis berries and Schisandra lignans showed varying activity on two different clinical isolates of C. pneumoniae, CV-6 and K7. The Schisandra lignans were shown to act at the mid-cycle of the infection. In addition, it was found that the effect is more targeted to the intracellular forms of the bacteria than to the extra cellular forms. Moreover, the results suggest that the antichlamydial mechanism of these lignans is specific to the host cell Chlamydia interaction rather than caused by their antioxidative properties. Schisandra extract and lignans did not affect the growth of three common respiratory tract bacterial pathogens, nor did they inhibit the growth of seven common metabolically active species, suggesting a Chlamydia-selective action of the extract and the lignans. Most of the lignans did not decrease host cell viability at the studied concentrations. As effective and selective growth inhibitors of Chlamydia pneumoniae, non-toxic and most of them not violating the Lipinski s rule of five, these Schisandra lignans present excellent lead candidates for further drug discovery. Schisandra lignans were utilized in constructing a reference set for a novel ligand-based virtual screening process, which proved successful with a hit rate of 1.2%. Six non-toxic lead compounds were found, belonging to new antichlamydial chemotypes. A new chemical space for antichlamydial compounds was defined, for future antichlamydial drug discovery projects.
  • Dimitrow, Sari Maarit (Helsingin yliopisto, 2016)
    Development and Validation of a Drug-Related Problem Risk Assessment Tool for Use by Practical Nurses Working With Community-Dwelling Aged The demand for long-term home health care services for the aged (≥65 years) is growing. Practical nurses (PNs) are those who most often visit the aged using HC services and consequently, are in a key position to monitor the benefits and risks of pharmacotherapy of their clients. The aim of this study was to develop and validate an easy-to-use DRP Risk Assessment Tool (DRP-RAT) for PNs caring for home-dwelling aged ≥65 years focusing on identifying and solving the highest priority DRP risks. The specific aims were: 1) to systematically review articles that describe criteria for assessing inappropriate prescribing in the aged ≥65 years; 2) to describe the development process and content validation of the DRP-RAT; 3) to evaluate the feasibility of the final DRP-RAT among PNs in HC; and 4) to assess the reliability of risk assessments conducted by PNs by using DRP-RAT and to identify the clinically most significant DRPs needing action. Two systematic literature reviews (Study I, year 2010; an unpublished one) and the expertise of the research group were used as a basis for the development of the DRP-RAT. The content of the draft tool was validated by a three-round Delphi survey with a panel of 18 experts in geriatric care and pharmacotherapy (Study II, year 2010). Data for the feasibility study were collected during the training of PNs, working in HC, in the use of the DRP-RAT (Study III, year 2011). The PN-conducted (n=25) DRP risk assessments by DRP-RAT (n=85) and the same clients copied medication lists (n=68), face-to-face discussions and responses to open questions of the returned feedback forms (n=23) were analyzed. In 2013, an experienced geriatrician reviewed HC clients (n=45) medications by using three different reviewing methods on each patient (Study IV). The methods based on: 1) DRP-RAT (n=45) completed by the PNs (n=26) and copied medication lists; 2) health centre s medical records ( gold standard ); and 3) Methods 1 and 2 together. Results of the reviews and contents of the geriatrician s open comments regarding the PNsʼ risk assessments were analyzed. DRPs in the study population identified and reported by the geriatrician were studied from the geriatrician s DRP classifications (n=45) and by a retrospective review of the geriatrician s case reports (n=45). The final DRP-RAT consists of 18 items that assess risks for DRPs in home-dwelling clients. It is divided into four sections: 1) Basic Client Data, 2) Potential Risks for DRPs in Medication Use, 3) Characteristics of the Clientʼs Care and Adherence and, 4) Recommendations for Actions to Resolve DRPs. The DRP-RAT turned out to be feasible among PNs and the PN-completed tool was capable of providing reliable and timely patient information to support physician s clinical decision making. Compared to the gold standard (Method 2), Method 1 resulted in a false negative rating in 7% (95 % CI 1.4 18.3) of the cases (3/45). The geriatrician identified an average of 3.1 potential DRPs per patient. This study indicates that the DRP-RAT, developed and validated in this study, could make it possible to more effectively involve PNs, working in HC, in medication risk management among the home-dwelling aged, and that medication risk management should be focused on the highest priority risks. Actions to facilitate the implementation of the DRP-RAT in the Finnish health care system are needed. Future studies are needed to evaluate the effects of PNsʼ risk assessments using the DRP-RAT on clinical, humanistic and economic outcomes.
  • Kuryk, Lukasz (Helsingin yliopisto, 2016)
    Despite major advances in conventional cancer treatments by surgery, chemotherapy, radiotherapy and their combination, the outcome remains partially ineffective against numerous cancer types, for example mesothelioma, lung cancer, and colon cancer. Furthermore, due to resistance factors and the subsequent loss of response, which may occur rapidly during the conventional treatments regimes, new anti-cancer agents, presenting new mechanisms of action and lacking cross-resistance to commonly used therapies, are in high demand. Oncolytic virotherapy is a promising anti-cancer strategy, and the approval of the first oncolytic virus, Imlygic (T-Vec, talimogene laherparepvec), in Western world by US Food and Drug Administration (FDA) and European Medicines Agency (EMA) has opened up new perspectives for improved treatment of cancer. Single therapy is rarely successful in treating cancer, particularly in metastatic or advanced cancer, and survival rates with monotherapies alone are generally poor. The combination of multiple therapies to treat cancer has already shown significant results in the standard care of cancer. This strategy utilizes the combination of both conventional and novel therapies that can bring the future promise of cancer treatment. In this thesis it has been hypothesized that by combining oncolytic adenoviruses (oAd) with chemotherapeutic drugs and a biological agent we could improve anti-cancer efficacy through synergistic effect against cancer. Therefore, we have tested various treatment regimes with the overall goal being the improvement of oncolytic virotherapy efficacy. Secondly, since safety issues concerning gene therapy and viral vectors are tremendously important, we have performed studies on safety issues of adenoviral vectors. In brief, we have evaluated the anti-cancer activity of combination treatment with standard of care (SoC) chemotherapy (Pemetrexed, Cisplatin, Carboplatin) and Ad5/3-d24-GM-CSF (ONCOS-102) in vitro and in a xenograft BALB/c model of human malignant mesothelioma (MM). We could show improved anti-tumor effects when ONCOS-102 was combined with SoC chemotherapy regimens over chemotherapy and virus alone. Combination therapy resulted in synergistic anti-cancer effect improving the therapeutic outcome. In a subsequent study we tested anti-cancer properties of the dipeptide L-Carnosine complexed with an oncolytic adenovirus (virus-L-Carnosine complex). The complex demonstrated improved anti-tumor efficacy both in vitro and in vivo in tested cancer models. In HCT116 colon and A549 lung cancer cells, the virus-L-Carnosine complex presented a higher transduction level and infectious titer over uncoated oncolytic adenovirus. The in vivo efficacy of the virus-L-Carnosine complex was tested in two cancer models: i) lung and ii) colon cancer xenograft mice models. It exhibited a significant reduction in tumor growth compared to other tested groups. Additionally, we investigated the molecular mechanism underlying the effects of the complex on tumor growth reduction. Safety assessment of viral vectors was performed in animal studies. Extensive studies on toxicity and bio-distribution of ONCOS-102 in Syrian hamsters and experiments in BALB/c nude mice indicated no side effects of repeated administration of oncolytic adenovirus. The side effects were evaluated by assessment of body weight, food consumption, hematology, clinical chemistry, histopathology and bio-distribution. We concluded that combinatory studies utilizing oncolytic viruses with standard of care chemotherapy and an experimental virus-L-Carnosine complex showed synergistic anti-cancer efficacy, thus providing a strong rationale for clinical testing of such combinations in mesothelioma, lung and colon cancer. Additionally, our studies suggested that adenovirus could be used in future studies for delivery of other bioactive drugs as a novel strategy in cancer therapy.
  • Malinovskaja-Gomez, Kristina (Helsingin yliopisto, 2016)
    Biological variation and poor transport efficacy are the major concerns in the development of novel iontophoretic drug delivery systems for the transdermal administration of therapeutics. One possibility to overcome these limitations would be to load the drug in interest into a reservoir system such as ion-exchange fibers or nanocarriers prior administration. More precise and homogenous control of drug release and the following transdermal iontophoretic permeation could be obtained as the transdermal device/patch would determine the rate of drug transfer instead of the skin, leading to smaller inter- and intrasubject variability. In addition, the range of molecules delivered by iontophoresis can be expanded as charges could be imparted to neutral drugs by encapsulating them in charged drug carriers. Other benefits raising from such combined systems include enhanced drug transport into or across the skin, improved drug stability and decreased local side effects on skin. The aim of this thesis was to study in vitro the applicability of systems that combine iontophoresis and either drug-loaded ion-exchange fibers or nanocarriers for the controlled transdermal delivery of therapeutics. Firstly, drug reservoirs based on cation-exchange fibers were utilized to retard drug release and provide additional control into transdermal transport of a small molecular drug and a peptide. The drug release kinetics could be modified by the choice of the fiber type or the ionic composition of the external solution. The application of pulsed current iontophoresis instead of conventional constant current led to increased transport efficiency of a cationic hydrophobic peptide that has a tendency to adsorb into skin and inhibit electroosmosis as its main transport mechanism. In addition, drug delivery systems combining iontophoresis and nanoencapsulation into polymeric nanoparticles or lipid vesicles for the controlled transdermal delivery of lipophilic or hydrophilic model compound were developed and tested. Although the obtained nanocarriers were considered as suitable for transdermal iontophoretic administration, regarding the colloidal properties, stability and release kinetics, no clear advantage was observed with respect to drug permeation from free drug formulation. Throughout the thesis, the impact of formulation parameters and current type on drug transport efficiency was monitored. Iontophoretic transdermal drug delivery from polymeric nanoparticle-based formulations but not from lipid vesicular nanocarriers was improved by the application of pulsed current. In conclusion, binding the drug molecules prior iontophoresis into reservoir based on ion-exchange material or nanocarriers is a promising approach to be utilized in controlled transdermal delivery, although the comprehensive evaluation of full potential of such systems tailored for specific drug warrants further investigation in the future.
  • Lajunen, Tatu (Helsingin yliopisto, 2016)
    Biomolecules are emerging as the most important source of new therapeutic compounds. Commonly these molecules are fairly large and unstable in biological environment. Furthermore, the target sites are often located inside cells and specialized tissues. Nanoparticle based systems, including liposomes, have become the most studied method of biologics delivery. They increase drug stability in blood circulation and facilitates drug accumulation at the target site. However, often the amount of drug released remains insufficient. Lately, several stimuli-responsive nanoparticles have been developed for better control of the drug release. Among these are light triggered liposomes, which are the focus of this work. Liposomes consist of spherical bilayer forming lipids, phospholipids and sometimes additional stabilizers, such as cholesterol. The liposomes in this thesis were made with different manufacturing processes, among which the most common was the thin film hydration method. The size of the formed liposomes was reduced by extrusion, sonication or high pressure microfluidization. Light triggered release of cargo from the liposomes was achieved by encapsulating gold nanoparticles (AuNP) or indocyanine green (ICG), that convert light energy into heat. The produced heat affects the thermosensitive bilayer of the liposomes, making it more permeable for the drug molecules. The fluidity of the bilayer was analyzed to determine the optimal phospholipid composition. The size of the liposomes was measured by dynamic light scattering to evaluate the size reduction and uniformity. The stability of the different formulations was evaluated and compared with each other. Fluorescent molecules were used as model drug compounds to study the release properties of the liposomes in controlled in vitro and cell experiments. In this work, a large portfolio of methods and formulations was developed. By combining these properties into a single drug delivery system, efficient protection of the cargo and the healthy tissue, distribution to challenging target sites, controlled spatial and temporal drug release can be achieved. The next steps in this work involve evaluation of the optimized carrier with applicable disease models, analysis of the in vivo pharmacokinetics and more profound toxicological experiments. Even though many challenges remains to be solved, the beneficial qualities of the light triggered liposomes show great potential for treatment of posterior eye conditions, cancer and other diseases lacking in therapeutic efficacy.
  • Antila, Hanna (Helsingin yliopisto, 2016)
    Major depressive disorder is one of the most significant causes of disability worldwide. Currently, the main treatment options for depression are psychotherapy and antidepressant drugs that pharmacologically target the monoamine systems such as serotonin transporter (SERT) blocker fluoxetine. It has been hypothesized that impairments in synaptic function and plasticity, caused for example by stress, could underlie the manifestation of depression. Moreover, in rodent models chronic treatment with antidepressant drugs has been shown to enhance plasticity of the adult brain via brain-derived neurotrophic factor (BDNF). The effects of BDNF mediated via its receptor tropomyosin receptor kinase B (TrkB) promote synapse function and thus could facilitate recovery from depression. Interestingly, antidepressant drugs with different main pharmacological targets seem to share the ability to activate the TrkB receptor, however, the mechanisms how antidepressant drugs activate TrkB are not known. The delayed onset of action and limited therapeutic efficacy of antidepressant drugs has promoted interest toward finding more rapid-acting and effective treatment options for depression. Electroconvulsive therapy has been the treatment of choice for treatment resistant depressed patients, however, side effects and the disrepute among general public has limited its use. Recently, subanesthetic doses of dissociative anesthetic ketamine have been shown to rapidly alleviate depression symptoms in depressed patients who do not respond to conventional antidepressant drugs. The effects of ketamine on mood appear already couple of hours after single intravenous infusion and last for about one week. Ketamine has been shown to induce mammalian target of rapamycin (mTOR) via BDNF-TrkB signaling, rapidly promote synaptogenesis and alter neural network function. Furthermore, in small human studies another anesthetic isoflurane has rapidly alleviated symptoms of depressed patients. Yet, the potential of isoflurane in the treatment of depression has not been studied in large clinical trials. Since TrkB receptor is involved in regulation of synaptic plasticity, drugs that act as agonists or positive allosteric modulators of TrkB could be potentially beneficial in the treatment of CNS disorders characterized by impaired plasticity. The first aim of our studies was to develop a platform suitable for high-throughput screening of compounds regulating TrkB activity. We developed an in situ ELISA (enzyme-linked immunosorbent assay) method that detects phosphorylated TrkB receptors from cultured cells. The main advantage of the in situ ELISA compared to conventional ELISA is that the cells are cultivated directly on the ELISA plate making the additional transfer step of the cellular material from the cell culture plate to the ELISA plate unnecessary. To further validate the in situ ELISA method, we conducted a proof-of-concept screening of a small chemical library and found several compounds that dose-dependently activated TrkB receptor or inhibited BDNF-induced TrkB activation. The second aim was to examine the mechanism how the antidepressant drugs activate TrkB. Interestingly, we found that antidepressant drugs activate TrkB independently of BDNF. Moreover, SERT, the main pharmacological target of fluoxetine, was not required for the fluoxetine-induced TrkB activation. Furthermore, the antidepressant-induced TrkB activation was developmentally regulated. The ability of antidepressants to activate TrkB appeared around postnatal day 12. Interestingly, at this same developmental timepoint (P12) the ability of BDNF to activate TrkB decreased dramatically. Finally, we aimed to characterize the neurobiological basis for the possible antidepressant effects of isoflurane. We found that brief isoflurane anesthesia rapidly and transiently activated the TrkB-mTOR signaling and produced antidepressant-like behavioral response in the forced swim test in a TrkB-dependent manner. Single isoflurane treatment also produced an antidepressant-like phenotype in behavioral paradigms that normally require chronic treatment with conventional antidepressant drugs, suggesting that isoflurane may have rapid antidepressant effects similar to ketamine. Moreover, isoflurane facilitated hippocampal long-term potentiation when measured 24 hours after the treatment and affected the general neural network function by increasing activity of the parvalbumin-positive inhibitory interneurons in the hippocampus. In conclusion, our results improve the understanding of the mechanism of action of conventional antidepressant drugs and provide plausible neurobiological basis for the antidepressant effects of isoflurane. Our findings also support examining further the potential of anesthetics in the treatment of depressed patients who do not respond to the current treatment options.
  • Stepniewski, Michal (Helsingin yliopisto, 2016)
    The cell membrane is a gateway to the cell and immersion point for membrane proteins and thus is of interest for pharmacology and structural biology. This thesis aims to study its interaction with water, small molecules, polymers and proteins through molecular dynamics simulation and statistical analysis. In the first part of the thesis, I have performed a statistical analysis of membrane proteins present in the PDB databank and enumerated in a structure database of known membrane proteins. Based on a statistical analysis of 127 proteins it was shown that extracellular cysteines are not solvent accessible. This rule has not previously been stated and was poorly followed by the participants of the GPCR DOCK competitions in 2008 and 2010. Thus it can provide qualitative guidelines to improve structural modeling. In a second study, based on a statistical analysis of 39 membrane proteins of three or more transmembrane helices, all of different fold, we have shown and clustered different spatial arrangements that sets of three interacting or consecutive helices can take, in addition to visualizing their abundance. In the second part of the thesis, I performed 200 ns simulations of both membranes in the gel (DSPC) and liquid-crystalline (DLPC) states with solvent and ions; These simulations were repeated with functionalized PEG polymers included (PEGylation). We also performed 200ns lipid membrane simulations in the liquid-crystalline (POPC) state with hematoporphyrin. Our studies provide a new, more accurate description of interactions between lipid membrane ions and featuring PEG polymers rather as dynamic molecules looping around Na+ ions and penetrating to liquid crystalline membrane rather than just a steric barrier outside of membrane. This sheds new light on the mechanism of liposome protection by PEG as well as triggering the release of liposome content through a heat induced lipid phase transition. Hematoporphyrin was shown to reside in the lipid headgroup carbonyl region. Ionized hematoporphyrin has lower affinity to the membrane as well as forming stable dimers in the aqueous phase. The research was in agreement with experimental data and has provided a molecular level view of the interactions between photosensitizers and the membrane.
  • Kekäle, Meri (Helsingin yliopisto, 2016)
    The establishment of tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of chronic myeloid leukemia (CML) during the last fifteen years. This study explored the journey of 86 patients with CML using oral TKI treatment in Finland. The aim was to assess their adherence to TKIs and how the adherence, which is crucial for treatment outcomes, could be improved. This study applied quantitative and qualitative methods and a randomized controlled study design during 2012-2014. All patients participated in the in-person interview, which followed the idea of the patient s journey with CML from the time before diagnosis to the study point. Patient-reported adherence was evaluated using Morisky s 8-Item Medication Adherence Scale (MMAS) (I-IV). Physicians were also asked to assess their patients adherence. Patient-reported adverse drug reactions (ADRs) and quality of life (QoL) were assessed during the interview using a structured questionnaire (II, III). The patient s knowledge of the disease and TKI treatment was evaluated by asking five key questions (I, III, IV). The intervention with 9-month follow-up in Study IV was based on tailored patient education combining nurse-conducted face-to-face counseling, educational video, patient booklet, website and text message reminders. The intervention material had the following learning objectives: CML as a disease, goals for TKI treatment, the importance of taking TKI medication as prescribed, and self-management of ADRs. A total of 86 patients participated in the study (approximately 20% of all Finnish CML patients). Of the patients enrolled, 43 were randomized into the intervention group and 43 into the control group (IV). A total of 68 patients completed the study. The results show that the response to TKI treatment was high (I), with 81% of the patients showing an optimal response to their treatment according to European LeukemiaNet 2013 recommendations. The CML patients knowledge of the disease and its treatment was poor (I). Despite the high molecular response rates to TKIs, adherence was not good in most of the patients: less than a quarter (23%) showed high adherence, 56% medium adherence, and 21% low adherence (I). Adherence was not influenced by patients gender, age, education, knowledge, time from diagnosis, ADRs, number of comorbidities or number of other medications. There was a considerable difference between observed and experienced adherence: 94% of the patients were highly adherent according to the physicians assessment, compared to 23% (I). The most common reason for unintentional non-adherence was forgetting to take the medication (I, III, IV). In the interviews patients reported self-regulation of medication taking, particularly on those occasions where patients wanted to avoid ADRs (III). The incidence of patient-reported ADRs was high (II). At the time of the study 97% of the patients reported suffering from at least one ADR, most commonly muscle soreness or cramp (80%), swelling of hands, legs, feet, or around the eyes (69%), and fatigue (50%). Patient interviews indicated that ADRs were the most common barriers to adherence (III). More than half of the patients felt the ADRs had a negative influence on their daily QoL (II). Compared with the total study group, the incidences of almost all symptoms were higher among patients whose symptoms negatively affected their daily life than those who reported no such influence (II). The patient journey model developed in the study (III) identified the following critical phases in the CML patient s journey: getting the diagnosis, starting the treatment, getting continuous support for treatment self-management, and managing fear caused by perceived severity of the disease. Even though only 44% of the low-adherent patients in the study experienced the TKI treatment as inconvenient, most of the patients (94%) were willing to stop taking the medication in the future if possible (III). All CML patients in the study were lacking a treatment plan and only a few had a medication list (I, III). The knowledge test (I, III) showed that patients had a poor understanding of their disease and its treatment, while low-adherent patients indicated that understanding the consequences of not taking the medication and the goal for the treatment would be motivating factors to adhere to the medication (III). The intervention significantly improved adherence (IV). In the intervention group the MMAS score increased more often than in the control group (p=0.001). The MMAS score declined in almost half of the patients in the control group, but only in 9% of those in the intervention group (p=0.001). A majority of the patients found the intervention useful, the most useful parts being face-to-face counseling and the educational booklet. Text messages were least valued. The findings of this study suggest that non-adherence is common among Finnish CML patients and that physicians seem to be too optimistic in assessing their patients adherence. The complex interplay between symptom burden, adherence, self-regulation, managing with ADRs, response to TKI therapy, and healthcare utilization highlights the need for regular symptom burden assessment in CML as a means to identify potential adherence problems before they affect the patients response to TKI treatment. Tailored patient education improved the adherence of patients with CML after a 9-month follow-up. Without the additional support, adherence behavior tended to decline. Patients were most satisfied with face-to-face counseling by the nurse, which means they need personal support and practical aids to help them manage their medication in everyday life. Access to personal counseling and information should be systematically planned as an essential part of CML care. Appropriate and updated information in printed and electronic formats should be available for nurses and other healthcare personnel to enable them to support their patients. The findings of this study suggest that patients perceptions and preferences should be understood and taken into account when designing patient education interventions for real-life clinical practice. The findings also highlight the need to further evaluate the interventions to enhance adherence. There is a need for communication to increase patients abilities to follow their treatment plan throughout their journey, which requires real partnership between healthcare professionals and patients.
  • Kopra, Jaakko (Helsingin yliopisto, 2016)
    Midbrain dopamine neurons exert a powerful influence on behavior and their dysfunction is associated with many neurological and neuropsychiatric diseases, including Parkinson s disease (PD). Dopamine neurons are large, complex and sensitive cells. Hence, their survival and correct function requires coordinated action of various transcription and regulatory factors both during development and aging. Potentially, one such factor is glial cell line-derived neurotrophic factor (GDNF). Ectopically applied GDNF is best known for its potent ability to protect and restore damaged dopaminergic neurons both in vitro and in vivo. GDNF-based therapies have been tested in clinical trials with PD patients with variable success. However, the function of endogenous GDNF in brain dopamine system development, aging and disease is poorly understood. Improvement in GDNF-based therapies requires better understanding of the physiological functions of GDNF in the brain. The current knowledge of endogenous GDNF function remains obscure, mainly due to the lack of proper animal models. The present study investigated the regulatory role of endogenous GDNF in the development, maintenance and function of midbrain dopamine neurons utilizing novel mouse models: GDNF conditional knock-out (cKO) mice and GDNF hypermorphic (GDNFh) mice over-expressing GDNF from the endogenous locus. GDNF cKO mice enable GDNF deletion solely from the central nervous system during embryonic development or later in adulthood, preserving its vital role in kidney development. Midbrain dopamine systems of these new mouse strains were studied with immunohistochemical, neurochemical, pharmacological, behavioral and molecular biology methods. We found more substantia nigra dopaminergic cells and elevated striatal dopamine levels in immature and adult GDNFh mice. In cKO mice, dopamine levels and cell numbers were unaltered, even upon aging, and regardless of the timing of GDNF deletion. Both mouse strains exhibited enhanced dopamine uptake, while responses to amphetamine were augmented in GDNFh mice and reduced in cKO mice. GDNFh mice also released more dopamine and GDNF elevation protected them in a lactacystin-based model of PD. Overall, dopamine neurons were more sensitive to moderate elevation than complete absence of endogenous GDNF, which suggests that they can adaptively compensate for GDNF loss. This highlights the limitation of broadly utilized gene deletion approaches in analyzing gene function. Our results indicate a clear role for endogenous GDNF in midbrain dopamine neuron development and function, but also demonstrate that GDNF is not required for their maintenance during aging. Furthermore, the ability of endogenous GDNF to protect animals in a PD model without the side effects associated with ectopic GDNF application suggests that elevation in endogenous GDNF levels may be an important future route for PD therapy.