Farmasian tiedekunta

 

Recent Submissions

  • 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.
  • Svanbäck, Sami (Helsingin yliopisto, 2016)
    Key physicochemical properties determining the developability of a drug include solubility, dissolution rate, lipophilicity and pKa. Not only do these properties affect synthesis and solid form optimization, choice of administration route, processability and formulation strategies; they also greatly influence, directly or indirectly, the absorption, distribution, metabolism, excretion, toxicity and efficacy of drugs. However, miniaturized methods that would enable small-scale determination of these fundamental properties in an accurate and rapid way, are lacking. Image-based microscopy could provide an opportune method for non-specific, rapid and miniaturized applications. First, the applicability of image-based microscopy and single-particle analysis in drug dissolution rate measurement was evaluated. This was done by comparing image analysis data with traditional UV spectrophotometric data of individual dissolving drug pellets. It was found that dissolution rates obtained by image analysis and UV spectrophotometry were practically identical. Next, a single-particle trap flow-through device was developed, wherein it is possible to continuously monitor individual drug particles under constant flow conditions. Based on the promising results of image-based dissolution rate analysis, the possibility of acquiring the intrinsic dissolution rate from individual freely rotating particles, trapped inside the flow through device, was evaluated. It was found that image analysis can be used for rapid real-time determination of intrinsic dissolution rates from continuously changing effective surface areas of dissolving individual micro-particles. The method was then further extended to determine the equilibrium solubility of drugs. Based on the diffusion layer dissolution rate model, solubility is the rate limiting factor of dissolution and can therefore be determined. While solubility is generally determined from bulk solutions after long incubation times, it was shown that the equilibrium solubility can be rapidly determined from individual pure-substance particles by means of the diffusion layer theory and image analysis. Finally, the single-particle method was further miniaturized and a second device developed, in order to allow imaging of individual powder crystals. It was shown that dissolution rate and solubility can be acquired from individual nanogram crystals. The single-particle method was further extended to acquire pKa, logP and logD of the studied substances, using aqueous buffers, simulated physiological solutions and organic solvents. Using this method and device, it is possible to acquire a complete pH-solubility profile for an unknown material of unknown composition, with individual measurements of less than 30 seconds. In summary, these results strongly suggest that image-based analysis of materials could be applied in high-throughput experimentation (HTE) applications. The possibility of acquiring solubility, dissolution rate, lipophilicity and pKa using a single analytical method, could significantly simplify and speed up accurate data acquisition. This in turn, could lead to faster and more informed decision-making and, ultimately, better and more affordable drugs.
  • Borrel, Alexandre (Helsingin yliopisto, 2016)
    This thesis presents the development of computational methods and tools using as input three-dimensional structures data of protein-ligand complexes. The tools are useful to mine, profile and predict data from protein-ligand complexes to improve the modeling and the understanding of the protein-ligand recognition. This thesis is divided into five sub-projects. In addition, unpublished results about positioning water molecules in binding pockets are also presented. I developed a statistical model, PockDrug, which combines three properties (hydrophobicity, geometry and aromaticity) to predict the druggability of protein pockets, with results that are not dependent on the pocket estimation methods. The performance of pockets estimated on apo or holo proteins is better than that previously reported in the literature (Publication I). PockDrug is made available through a web server, PockDrug-Server (http://pockdrug.rpbs.univ-paris-diderot.fr), which additionally includes many tools for protein pocket analysis and characterization (Publication II). I developed a customizable computational workflow based on the superimposition of homologous proteins to mine the structural replacements of functional groups in the Protein Data Bank (PDB). Applied to phosphate groups, we identified a surprisingly high number of phosphate non-polar replacements as well as some mechanisms allowing positively charged replacements. In addition, we observed that ligands adopted a U-shape conformation at nucleotide binding pockets across phylogenetically unrelated proteins (Publication III). I investigated the prevalence of salt bridges at protein-ligand complexes in the PDB for five basic functional groups. The prevalence ranges from around 70% for guanidinium to 16% for tertiary ammonium cations, in this latter case appearing to be connected to a smaller volume available for interacting groups. In the absence of strong carboxylate-mediated salt bridges, the environment around the basic functional groups studied appeared enriched in functional groups with acidic properties such as hydroxyl, phenol groups or water molecules (Publication IV). I developed a tool that allows the analysis of binding poses obtained by docking. The tool compares a set of docked ligands to a reference bound ligand (may be different molecule) and provides a graphic output that plots the shape overlap and a Jaccard score based on comparison of molecular interaction fingerprints. The tool was applied to analyse the docking poses of active ligands at the orexin-1 and orexin-2 receptors found as a result of a combined virtual and experimental screen (Publication V). The review of literature focusses on protein-ligand recognition, presenting different concepts and current challenges in drug discovery.
  • Kanninen, Liisa (Helsingin yliopisto, 2016)
    Standard two-dimensional (2D) in vitro cell culture systems do not mimic the complexity found in the liver as three-dimensional (3D) cell-cell and cell-matrix interactions are missing. Although the concept of cell culturing was established over 100 years ago the currently used culture techniques are not yet ideal. In the field of pharmacy especially, the need of physiologically-relevant models to characterize biotransformation pathways during drug development is urgent. Hepatocytes, the main cell type of the liver, are essential components in these in vitro models. Liver cell lines and derivation of hepatocyte-like cells from stem cells are alternative sources to primary isolations for obtaining hepatocytes. In the liver, hepatocytes are in continuous interaction with other cells and surrounding extracellular matrix (ECM). Moreover, liver functions are strictly dependent on correct tissue architecture. One approach to improve the standard cell culture systems is to mimic the hepatocytes natural microenvironment and organization by culturing the cells within biomaterial matrices. Matrix-based culture systems for hepatocytes have been developed from natural, synthetic and hybrid biomaterials and the cells can be grown in 2D or 3D configuration. The aim of this thesis was to find new defined culture matrices for in vitro hepatic differentiation. First, we studied two biomaterials, nanofibrillar cellulose (NFC) hydrogel and hyaluronic acid-gelatin (HG) hydrogel, to construct functional liver 3D organoids. Both of the studied hydrogels supported 3D spheroid organization of human liver progenitor HepaRG cells and their functional polarization. The 3D culture systems promoted hepatic differentiation of progenitor cells faster than the standard 2D culture. However, the 3D hydrogels did not enhance hepatocyte-like properties if the HepaRG cells were pre-differentiated to hepatocyte-like cells in advance. Subsequently, we showed that NFC hydrogel culture can be combined with high-resolution imaging since the intact spheroids can be enzymatically released from the matrix. This was not possible with the HG hydrogel. We demonstrated that silica bioreplication preserved the 3D spheroid structure with its fine details and cellular antigens and allowed detailed morphological analysis of the spheroids cultured in NFC hydrogel. Next, we developed a xeno-free matrix for hepatic specification of human pluripotent stem cell-derived definite endoderm (DE) cells using a three-step approach. We first proved our hypothesis that a liver progenitor-like matrix, HepaRG-derived acellular matrix (ACM), supports hepatic lineage differentiation of DE cells. Then, we characterized the ECM proteins secreted by HepaRG cells, and finally we showed that the identified proteins, laminin-511 and laminin-521, can replicate the effect of HepaRG-ACM. The human pluripotent stem cell-derived hepatic cells expressed mature hepatocyte-like functions but the phenotype of the cells was eventually closer to fetal hepatocytes than mature cells. Thus, hepatic maturation should be further studied. In conclusion, this thesis describes new biomaterials for hepatic differentiation, a protocol to form 3D spheroids and to transfer intact spheroids to high-resolution imaging, and that the described three-step approach can guide the identification of new defined matrices.
  • Herranz Blanco, Bárbara (Helsingin yliopisto, 2016)
    Drug delivery systems (DDS) have been developed in the last decades to improve the pharmacological properties of free drugs by modifying their pharmacokinetic profile and biodistribution. Major limitations for newly developed drug molecules are the poor water solubility and stability, which can be addressed by DDS. These can protect the drugs from the potentially harsh external conditions found in the biological fluids, and improve their dissolution rate by different strategies, overall increasing the therapeutic activity of the drugs. Additionally, chemotherapeutic agents are nonspecific in nature, leading to deleterious off-target side effects, and poor therapeutic efficacy. Therefore, targeted therapy plays a very important role in cancer treatment, although not without obstacles, since DDS have to overcome a number of biological barriers following their intravenous administration, including renal clearance or opsonization-mediated phagocytosis and efficient extravasation to the tumor. Mesoporous silicon (PSi) micro- and nanoparticles offer numerous benefits for biomedical applications, in particular for drug delivery. Along with a great biocompatibility and biodegradability, PSi possess mesopores (2‒50 nm), where the drugs can be easily loaded and confined in their amorphous state avoiding extensive crystallization, thus, increasing their dissolution rate. However, the release of drugs from this platform is uncontrolled and fast, necessitating the use of strategies to tune the drug release. In this thesis, multiple approaches were used for the design and fabrication of hybrid composites for drug delivery and cancer therapy, including PSi and polymer‒drug conjugate-based DDS produced by different modalities of the microfluidics technology and pH-switch nanoprecipitation. First, the loading and release of drugs with different solubility characteristics from PSi were investigated, and further PSi-lipid and polymer-composites were developed to control the drug release profiles. Overall, it was achieved both a sustained release of hydrophilic and hydrophobic molecules loaded on the PSi and also a reduced initial burst release from the bare PSi particles. Next, PSi-based nanovectors were envisaged for antitumoral applications. A smart PSi-based hybrid nanocomposite with stealth properties was developed, consisting of a pH-responsive polymeric structure assembled on the surface of drug-loaded PSi nanoparticles. This nanocomposite was extremely efficient avoiding drug release from PSi under physiological conditions, while allowing the release of the drug upon acidification of the medium. Remarkably, the nanocomposites avoided extensive macrophage recognition and phagocytosis. Thereupon, a tumor targeted theranostic nanoplatform with dual pH- and magnetic-response capacity was designed. The DDS consisted of a polymeric-drug conjugate nanoparticle containing an imaging agent and decorated with a tumor homing peptide for targeted drug delivery, which was successfully applied for intracellular triggered drug release. Overall, the hybrid composites based on PSi and a polymer-drug conjugate represented an advanced contribution to the field of drug delivery and cancer therapy, and in particular to the development of PSi as a platform for advanced drug delivery applications.
  • Shrestha, Neha (Helsingin yliopisto, 2016)
    Regardless of the considerable efforts, there have been no major breakthroughs in the development of effective oral protein/peptide delivery. When compared to parenteral administration, oral delivery can significantly improve the patients quality of life, especially in chronic conditions, such as diabetes mellitus (DM), which requires multiple injections daily. However, oral absorption of proteins/peptides is severely limited by their physico-chemical properties and various physiological barriers in the gastrointestinal tract. Porous silicon (PSi) has emerged as a promising drug delivery system, owing to its beneficial properties, such as top-down production, customizable particle and pore morphology, easy surface modification, simple drug loading, biodegradability and biocompatibility. Thus, the aim of this dissertation was to develop a multifunctional PSi based platforms that would be able to overcome the physiological barriers and efficiently deliver insulin and glucagon-like peptide-1 (GLP-1) orally. First, the influence of different PSi surface chemistries was evaluated on the intestinal transport of insulin. Due to the negatively charged surface of PSi, there was minimal interactions with the intestinal cells. Thus, chitosan, a polycationic mucoadhesive biopolymer with permeation enhancing effect, was used to modify the surface of the PSi microparticles. When comparing different surface modification techniques, chemical conjugation of chitosan to PSi exhibited strongest cellular interaction, and the highest insulin permeation and uptake across the intestinal cell monolayers. Secondly, three different nanoparticles (NPs) were developed based on lipids, polymers and PSi, with and without chitosan coating, and evaluated as potential oral GLP-1 delivery system. The results showed that the chitosan-modified PSi NPs were the most efficient nanosystem with the best loading degree and the highest GLP-1 permeation across the cellular monolayer. To overcome several physiological barriers, the next step was to develop a multistage nanocomposite comprising of chitosan-conjugated PSi NPs that were coated with a pH responsive polymer, in order to deliver GLP-1 and dipeptidylpeptidase-4 (DPP4) inhibitor simultaneously via the oral route. This multistage nanosystem showed enhanced GLP-1 transport across the intestinal cell monolayers and across the rat intestinal tissue. Furthermore, the nanosystem also demonstrated hypoglycemic effect in vivo after the oral administration in diabetic rats. The efficacy of the nanosystem could be attributed to the combined effect of the permeation enhancing chitosan-modified PSi NPs, the presence of DPP4 inhibitor that prevented GLP-1 degradation, and the pH responsive coating that helped in avoiding premature GLP-1 release/degradation in the stomach. Moreover, it was shown that the mucoadhesivity and permeation enhancing ability of chitosan-modified PSi NPs could be significantly increased by further surface modification of NPs with either L-cysteine or cell-penetrating peptide (CPP). It was disclosed that electrostatic interactions between the NPs and the glycocalyx were the most prominent pathway for the transport and uptake of insulin from the NPs, together with the contribution of active transport, adsorptive endocytosis and clathrin-mediated endocytosis. Overall, advanced PSi-based systems were developed which successfully overcame several limitations associated with the oral delivery of biomacromolecules, and thus, showed high clinical potential as oral protein/peptide delivery systems for DM therapy
  • Hirvinen, Mari (Helsingin yliopisto, 2016)
    Cancer is the leading cause of death worldwide creating a need for novel cancer treatments that are more efficient but also safer and more specific. Oncolytic viruses (OVs) have shown a solid safety profile in clinical trials. OVs are nowadays considered immunotherapies because of to their ability to stimulate the host immune system to fight against cancer. Promising efficacy has been seen in some trials, however, efficacy is often seen only in a small group of patients. The purpose of the thesis was to improve the efficacy of OV therapies by boosting the immunogenicity of the viruses, and to optimize the therapeutic efficacy by selecting favorable patient populations and by developing a method to tailor the drug individually for each patient. In the first study, an oncolytic adenovirus (OAd) was modified to express human tumor necrosis factor alpha (hTNFα), a potent immunomodulatory cytokine. The TNFα-virus showed effective tumor cell killing associated with signs of immunogenic cell death and enhanced recruitment of immune cells to the infection site. We also saw potential for combining the TNFα-virus therapy with radiation. In another study the immunogenicity of an oncolytic vaccinia virus was enhanced by modifying it to express DNA-dependent activator of interferon-regulatory factors (DAI), a potent inducer of innate immune responses during virus infection. We showed that the DAI-virus induces expression of genes involved in immune responses, and treatments with the virus showed improved cancer-killing efficacy and immunogenicity in murine and human melanoma models, suggesting applicability also in vaccine design. Response rates after virotherapies vary between patients, and there is a lack of markers that would help predict the patient cohorts who would benefit from the therapy. We screened over 200 cancer patients treated with OAds for two Fc gamma receptor (FcγR) polymorphisms to determine if these polymorphisms would affect the responsiveness to the treatments. We observed a certain FcγR genotype combination (FcγRIIIa-VV + FcγRIIa-HR) to be predictive of poor overall survival after OAd treatments. To tailor the OV therapy for enhanced specificity, we developed a novel platform (PeptiCRAd) to coat a virus with tumor-specific antigens (peptides) for improved induction of cancer-specific immunity. Efficacy and immunogenic potency of the PeptiCRAd were shown in several in vivo models. Our results suggest that administration of tumor-specific peptides on the surface of OVs increases the anti-tumor efficacy compared to treatments with viruses or peptides alone. This platform has potential to be used as a carrier and adjuvant for patient-specific peptides to trigger anti-tumor immunity in a personalized manner.
  • Montalvão, Sofia (Helsingin yliopisto, 2016)
    Marine environment is prolific in organisms with unique properties. Seas and oceans contain a wide diversity of species with biologically active metabolites that represent a valuable source with great potential for the development of novel therapeutic agents. This dissertation is focused on the biological study of synthetic compounds based on marine scaffolds as well as on marine natural product extracts originating from the Aegean Sea. Furthermore, it offers an introduction on the importance of marine natural products in the search of new bioactive compounds, the use of natural products as scaffolds for the synthesis of new drugs, and a general overview on bioactivity screening and the current status of marine-derived bioactive compounds as therapeutic agents. The potential of oroidin and clathrodin as parent structures for synthesis of novel compounds was explored. Antimicrobial and antiproliferative studies were conducted and it was concluded that 4-phenyl-2-aminoimidazoles 6g(I) and 6h(I) showed the best antimicrobial effect against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus), while compound 6j(I) showed the most interesting IC50 in antiproliferative studies. Compound 7(II), a synthetic derivative of 2-aminobenzothiazole, showed IC50 of 16 μM and 71 μM against a cancer cell line and a normal cell line, respectively. The selectivity index showed selectivity towards cancerous cells. In addition, okadaic acid was used as inspiration for the synthesis of crown ether acyl compounds. Compound (1,4,7,10,13,16-hexaoxacyclooctadecan-2-yl)methyl 3-(pyren-1-yl)propanoate) 1o(III) was found to be the most active in antimicrobial studies against Gram-positive Staphylococcus aureus with a MIC50 of 7.2 μM. The importance of bioprospecting the rich marine biodiversity in the Aegean Sea was also studied in this thesis. Biological activities of extracts from cyanobacteria, micro- and macroalgae were evaluated, and microalgae extracts (Amphora cf capitellata and Nitzschia communis) showed the most interesting antimicrobial results against Staphylococcus aureus and fungus Candida albicans. The results of the biological studies conducted in this thesis demonstrated antimicrobial and antiproliferative activity of several marine natural products and their synthetic derivatives. Further studies and structural optimization should be done to fully explore their potential for the development of therapeutic agents.
  • Kapp, Karmen (Helsingin yliopisto, 2015)
    Mentha plants are used in pharmaceutical and food industries. The genus Mentha has been classified into 18 species, 11 hybrids and hundreds of subspecies, varieties and cultivars. The medicinal and culinary properties of Mentha plants are bounded to the composition of polyphenolic compounds and essential oils. In the present study, the polyphenolic and essential oil composition of 47 Mentha plants. Similar profiling was conducted for 27 commercial peppermint tea samples. This was the first more detailed study to present the composition of mint flavourings isolated from 45 candies and food supplements. For the first time, the effects of peppermint teas water extracts, mint flavouring hydrodistilled extracts and terpenoidic reference substances against respiratory tract pathogen Chlamydia pneumoniae were investigated in vitro. Mentha plants water extracts and essential oils, mint flavouring hydrodistilled extracts and terpenoidic reference substances were tested in vitro for their antimicrobial properties against the potential pathogens Escherichia coli and Staphylococcus aureus. The antimicrobial effects of M. × villosa Huds., M. suaveolens Ehrh., M. × gracilis Sole, M. arvensis L. water extracts and mint flavouring hydrodistilled extracts were studied for the first time on E. coli and S. aureus. The polyphenolics of Mentha plants and peppermint teas water extracts were rather similar. The most abundant compounds found were rosmarinic acid and eriocitrin. Nine polyphenolic compounds were detected for the first time in the plants. The total content and composition of the essential oils in the Mentha plants and commercial peppermint teas samples varied. The essential oil analyses of peppermint teas showed that three tea samples may contain M. spicata L., different from that claimed on the package. The isolated mint flavouring hydrodistilled extract content was higher in mint flavoured sugar candies, pastilles and tablets than in chocolates. The three most abundant flavouring compounds were limonene, menthol and menthone. Peppermint teas water extracts were active against C. pneumoniae. The antichlamydial activity was often related to high content of luteolin and apigenin glycosides. The mint flavouring hydrodistilled extracts and reference substances decreased the infectivity of C. pneumoniae elementary bodies. Antichlamydial activity could be related to the high menthol content in extracts. Mentha plants water extracts showed antibacterial activity against S. aureus. Essential oils and mint flavouring hydrodistilled extracts inhibited the growth of E. coli and S. aureus. Linalool acetate and (-)-carvone were the most active reference substances against both bacteria. This study showed that consumption of Mentha plants may be beneficial for human health.
  • Matikainen, Minna (Helsingin yliopisto, 2015)
    The pharmaceutical industry has a vital reliance on successful new product launches (NPL), which are a critical driver of a company s performance. The prevailing literature on NPLs is fragmented and has mainly concentrated on a product s superiority as well as the strategic and tactical launch activities largely omitting the importance of customer relationships. The aim of this thesis is to provide a comprehensive overview on the key determinants of a successful NPL in the Finnish pharmaceutical industry. In practice, this study considers the extent to which a NPL and getting physicians to prescribe a new drug is relational activity. The role and relative impact of a company s strategic orientations and their mediating mechanisms were studied with survey data collected from the pharmaceutical companies operating in Finland. Partial least squares (PLS) path modeling revealed that the relationship orientation had the strongest positive impact on both customer acceptance and financial launch success. The company s accumulated market-based assets represented an alternative mediator in addition to product advantage. Sales force management and relationship marketing activities transformed a relationship-oriented organizational culture into launch performance. PLS regression modeling combined with target projection identified the diversity of determinants affecting launch performance at different stages of NPL. Product advantage and relationship marketing activities contributed to gaining the acceptance of key opinion leaders in the early phase of launch, while market-based assets and a company s relationship orientation largely determined the acceptance of the majority of target customers in the later phase. The buyer s perspective focused on the physician-pharmaceutical industry relationship and was studied by means of theme-interviews among a randomized sample of physicians. The positive relationship orientation of the physicians towards the pharmaceutical industry and whether they actively interacted with pharmaceutical companies were reflected in their early adoption of new drugs, especially when a product had a unique advantage and the physician s own personal interest accelerated the adoption of a new drug. In comparison, physicians who were negatively oriented towards the pharmaceutical industry and interacted passively adopted a new drug later based on evidence- and experience-based reasoning and the opinions of colleagues. In conclusion, this thesis calls for a relationship approach in order to complement the traditional sales and marketing approach regarding the launch of new pharmaceutical products. A successful pharmaceutical product launch should focus on appropriate relationship marketing activities that are conducted in a timely manner to achieve customer acceptance and financial launch performance.
  • Haavikko, Raisa (Helsingin yliopisto, 2015)
    Betulin is a triterpene class natural product present, for example, in the outer layer of birch bark, which is a low-value waste product of the forestry industry in Northern Hemisphere. Oxidation products of betulin, namely betulinic acid and betulonic acid, have been shown to have several biological activities. This makes betulin an interesting starting point for drug discovery projects. In this work, mainly ring A-fused heterocyclic derivatives of betulin were synthesized, and their structure-activity relationships against a protozoan parasite Leishmania donovani, prostate cancer cell lines, a serine hydrolase enzyme (ABHD12), and inflammatory factors were studied. The heterocycles used included pyridine, pyrazine, pyrazole, indole, isoxazole, and oxazole rings. Also, positions C-28 and C-20 of these compounds were modified and the resulting structure-activity relationships (SAR) were studied. Among the tested compounds, two heterocyclic betulin derivatives showed significant inhibition against L. donovani amastigotes growth. These compounds showed improved activity compared to the heterocyclic betulin derivatives tested previously. Also, a betulinderived potent anti-HIV compound, bevirimat, showed L. donovani growth inhibition at a similar level to the ring A-fused heterocyclic betulin derivatives. However, further optimization is needed to get more potent betulin derivative activity against L. donovani. In prostate cancer studies some of the betulin derivatives displayed dose-dependent anti-invasive activity at nanomolar concentrations with negligible cytotoxicity. The most potent compounds were betulin derivatives with a ring A-fused heterocycle. Also, the carboxyl group at C-28 seemed to be important for activity. These compounds showed considerably improved anti-invasive effects compared to betulinic acid. Betulin derivatives were found to selectively inhibit the ABHD12 serine hydrolase enzyme without inhibiting other endocannabinoid hydrolases and without activity towards cannabinoid receptors. In mechanistic studies, the inhibition type was shown to be reversible. Important structural features required for ABHD12 inhibition were revealed based on our SAR studies. Heterocyclic betulin derivatives were shown to suppress the expression of several inflammation mediators, such as iNOS, IL-6, and MCP-1, a pyrazole derivative being the most potent compound. With further improvement and development, more potent betulin derivatives could be found. The betulin derivatives show different activity with different targets, which means they are selective. Selectivity and potency could be further improved to obtain a potential betulin-derived compound for further development. In this work, betulin has been shown to be a good starting point for several different drug discovery projects.
  • Savolainen, Mari (Helsingin yliopisto, 2015)
    Parkinson's disease (PD) is characterized by a slow and gradual loss of neurons and subsequent loss of neurotransmitter dopamine in the movement-related nigrostriatal brain tracts. This causes symptoms of PD, which are resting tremor, rigidity and slowness of movement. Genetic and environmental factors are linked to PD, but its etiology is still largely unknown. Current drug therapies can only relieve the symptoms of PD but treatment to stop or delay the disease progression does not exist at the moment. The main findings in neuropathological characterization of the brain regions that are influenced in PD, are abnormal intraneuronal protein inclusions, called Lewy bodies. Insoluble, aggregated α-synuclein (aSyn) protein is the most abundant component of Lewy bodies. Pathological aggregation and accumulation of aSyn is associated with neuronal death in PD. Therefore, approaches to target aSyn as a potential disease-modifying treatment for PD have been under investigation. The aim of this study was to examine the role of prolyl oligopeptidase (PREP) and pharmacological inhibition of its enzymatic activity by KYP-2047 in aSyn aggregation. Using purified proteins and cell culture model, we showed that PREP forms direct protein-protein interaction with aSyn, thus enhancing its aggregation. KYP-2047 reduced the PREP-mediated aSyn aggregation in cell culture. Then the effects of PREP inhibition on aSyn aggregation were examined in vivo. The purpose was to first characterize a genetic mouse model, carrying mouse A30P mutated aSyn, which is linked to early-onset PD in humans, in order to find out if the mutated mouse aSyn is more prone to aggregate; and to cause PD-like phenotype. The effects of KYP-2047 treatments were assessed in the model. The main findings were that the A30P mutation in mouse aSyn protein caused minor hyperactive behaviour but did not change the brain dopamine levels, and A30P aSyn accumulated in the brain more than wildtype aSyn by age. KYP-2047 treatment reduced the amount of A30P aSyn in immunohistochemical analysis, and the reduction was more specific for high-molecular weight aSyn oligomers in Western blot analysis. We also observed increased autophagy markers in brain tissue. Therefore, PREP inhibition was further studied in cell culture, where it was shown to enhance macroautophagic protein clearance pathway, which is an important pathway in the degradation of high-molecular weight aSyn forms. In the last study, the effects of KYP-2047 were examined in a mouse model of PD that was based on lactacystin-induced inhibition of ubiquitin-proteasome protein degradation pathway. Proteasome inhibition was shown to induce rapid PD-like neurodegeneration recapitulating the cardinal features of PD. KYP-2047 treatment partially protected dopaminergic neurons in the brain and had beneficial effect on motor behaviour, but did not have an effect on aSyn amount. Taken together, this study has provided new insights into the role of PREP in aSyn aggregation and suggest that PREP inhibition has beneficial effects on reducing the aggregation process via two mechanisms. PREP inhibition could be promising and further assessed in the treatment of PD, other α-synucleinopathies and possibly other protein accumulation diseases.