Farmasian tiedekunta


Recent Submissions

  • Leino, Teppo (Helsingin yliopisto, 2018)
    Orexin peptides, orexin-A and orexin-B, and orexin receptor 1 and orexin receptor 2 form the orexin signaling system. The most studied part of the orexin system is its key role in sleep-wake regulation, although it is linked also to other physiological functions, such as addiction and nociception. To date, a large number of orexin receptor antagonists have been developed with one, suvorexant, having reached the market in the treatment of insomnia. However, much less attention has been paid to the development of small-molecular agonists of the orexin receptors, and only a few are known in the literature. Agonists might be beneficial for patients with narcolepsy or certain types of cancers. The aim of this thesis was to develop small molecules based on the azulene scaffold for targeting orexin receptors. Azulene is an unexplored ring structure in medicinal chemistry, however, it resembles other bicyclic aromatics such as indole and naphthalene, which are frequently found in drug molecules. The small number of existing general synthetic routes for azulenes possessing three or more substituents has most likely hindered the use of azulene-based compounds in medicinal chemistry. Due to this, the study was initiated by developing two different synthetic routes to access 1,3,6-trisubstituted azulenes. In the developed methods, the azulene scaffold was first synthesized from simple, readily available and inexpensive starting materials. Then the scaffold was functionalized via versatile synthetic handles, such as a halogen atom or a formyl group, which allow a facile generation of compound series. The efficiency of the synthetic routes was demonstrated with test substances, which gave good overall yields. The developed methods were used in the synthesis of azulene-based compounds, whose biological activity was assessed on the orexin receptors. The first series of compounds was based on the results from virtual screening of the library of 70 000 synthetically accessible azulene-based compounds. The second series was designed based on the results from the biological evaluation of the first series. With this approach, novel azulene-based ligands for orexin receptors were identified. The two most promising binders had Ki values in the low micromolar range and five other compounds acted as weak orexin receptor agonists. In addition, compounds potentiating the response of orexin-A to OX1 receptors in a concentration-dependent manner were discovered. These novel azulene-based compounds offer an interesting starting point for further development of antagonists, agonists and potentiators for orexin receptors.
  • Paukkonen, Heli (Helsingin yliopisto, 2018)
    The main role of excipients is to ensure the safety and efficacy of the whole pharmaceutical formulation throughout its shelf-life and administration. Formulation design and development as well as material testing are the key components for successful drug delivery. This is becoming increasingly complicated as new active pharmaceutical ingredients typically have poor solubility and/or bioavailability. Due to this, there is an ever increasing need to explore new excipients and material combinations as innovative formulation solutions are required. Furthermore, modified release formulations are needed to control the release rates and to adjust the desired therapeutic effects, raising even more demand for effective formulations. The main aim of this thesis was to evaluate the performance of plant based materials nanofibrillar cellulose (NFC) and anionic carboxylated nanofibrillar cellulose (ANFC) as pharmaceutical excipients for modified release formulations and bioadhesive films. These materials are widely available from renewable sources; biocompatible with relatively low toxicity combined with high mechanical strength and large surface area available for encapsulation. NFC and ANFC, together with HFBII protein, were used as emulsion stabilizers for encapsulation and release of poorly water-soluble drugs. The synergistic stabilization mechanism achieved with these biopolymers improved emulsions stability with extremely low concentrations. In another study, ANFC hydrogels were evaluated as matrix reservoirs for diffusion controlled drug release. Their rheological and drug release properties were shown to be preserved after freeze-drying and reconstruction. The ANFC hydrogels controlled the release kinetics of small molecular weight drugs moderately, whereas significant control was obtained in the case of large proteins. In a comparative study, three new grades of microcrystalline cellulose (MCC) hydrogels were evaluated for diffusion controlled drug release. MCC matrices efficiently controlled the release of both large and small compounds, indicating great potential for drug release applications in a similar manner to the ANFC hydrogels. Bioadhesive NFC and ANFC based films were prepared by incorporating bioadhesive polymers mucin, pectin and chitosan into the film structure. The bioadhesive properties of the films combined with good mechanical and hydration properties, together with low toxicity makes them a feasible option for buccal drug delivery applications. In conclusion, NFC and ANFC were shown to be versatile excipients applicable for several types of dosage forms. In the future, it is seen that these materials may be used systematically as functional excipients for modified release dosage form.
  • Laurén, Patrick (Helsingin yliopisto, 2018)
    Hydrogels are emerging as an important source for current biomaterial design, as they often possess intrinsic physical and mechanical similarities with soft tissue, are non-toxic and biocompatible. However, many hydrogel-based biomimetic materials are either derived from limited sources, or require external activators to achieve functionality, such as chemical crosslinking or environmental cues. Furthermore, many cross-linkers used with hydrogels are toxic, and environmental cues invoke slow responses. Therefore, to function as a rational biomaterial design for a biomedical application, these properties are preferably avoided, or improved with a composite system containing two or more polymer components to overcome these limitations. Plant-derived nanofibrillar cellulose (NFC) possesses the same intrinsic properties as many other hydrogels derived from the components of extracellular matrices (ECM). Therefore, NFC shares the biocompatibility and non-toxicity aspects of biomimetic materials. However, additional features of NFC can be exploited, such as shear-thinning properties, spontaneous self-gelation and chemical modification capabilities. Additionally, the source of NFC is practically inexhaustible, and is environmentally biodegradable, bearing no ecological burden. Therefore, when designing hydrogel-based biomaterials, NFC offers versatility, which enables the fabrication of potential biomedical applications for various purposes in an environmentally safe way. In this thesis, a wide range of potential applications of NFC-based hydrogels were investigated. These include 3D cell culturing, in vivo implantation and coating systems for drug and cell delivery, controlled drug delivery and local delivery as a bioadhesive system. These methods offer insight into the versatility of NFC-based hydrogels, which could improve the future design of biomaterials, for a safer and more efficient use in biomedical applications.
  • Capasso, Cristian (Helsingin yliopisto, 2018)
    With 1 685 210 new cases in 2016 in United States (American Cancer Society), cancer poses a serious challenge for the patients, the healthcare system and families of patients considering that 595 690 people will die from the disease. In the last decades, a new class of therapies raised particular interest thanks to the ability to promote long-lasting complete responses. Immune therapy exploits the patient´s own immune system to find and kill tumor cells. Once immunological memory is established, this can potentially grant long term protection. Nevertheless, the results of immunotherapies are often inconsistent as we observe a number of complete remissions together with patient that completely fail to respond to the therapy. Uncovering the mechanisms of such difference is the primary aim of researchers that try to optimize the strategies to ultimately increase the number of responders. In this thesis three main strategies will be reviewed, studied and finally combined: i) oncolytic adenoviruses; ii) cancer vaccines and iii) immune checkpoint inhibitors. In Study I, we developed an innovative cancer vaccine platform based on oncolytic adenoviruses. We used the adenovirus as a carrier able to deliver peptides to antigen presenting cells. The Virus-peptide complex (i.e. PeptiCRAd) was build with electrostatic interactions, thus preserving the virus ability to infect and kill tumors cells. In addition, we showed that PeptiCRAd was able to reduce the growth of B16OVA and B16F10 tumors by inducing antigen specific responses through the activation of dendritic cells. Ultimately, we showed its potential in humanized mice engrafted with both human melanomas and human peripheral blood mononuclear cells (PBMCs). In Study II we addressed the problem of optimizing the peptides formulations to be used in cancer vaccines. We set up an in silico framework that uses multiple in silico tools such as MHC-I affinity and immunogenicity predictions. This allowed us to directly compare how mutating specific residues would not only change the orientation of the peptide within the MHC-binding pocket, but it would re-shape the whole portion of the MHC molecule that is recognized by the TCRs. This gave us suggestions on the differences in anti-tumor efficacy observed when testing several several improved tumore-specific peptides against both B16OVA and B16F10 tumors. In Study III we used oncolytic vaccines to boost the response to PD-L1 blockade. The Combination proved to increase the survival of melanoma-bearing mice, promoting the presence of TILs with a non-exhausted state (PD-1+ TIM-3-). Hence checkpoint inhibition well synergized with the immune activating properties of the oncolytic vaccine. The findings described in this thesis highlight the necessity of combination therapies to increase the number of responders to immunotherapy. This is a critical aspect to enlarge the number of responding patients and to maximize the benefits of immunotherapies, considering that immunotherapeutic approaches are reaching the market with unprecedented speed.
  • Rimpelä, Anna-Kaisa (Helsingin yliopisto, 2018)
    Diseases of the posterior segment of the eye, such as age-related macular degeneration and diabetic retinopathy, are the leading causes of blindness in the developed world. Drug delivery to the posterior eye tissues, including the retina and choroid, is accomplished by frequent intravitreal injections, which are expensive and burdensome to the health care providers and patients, and may cause harmful side effects. Therefore, sustained and less invasive delivery strategies are vitally needed to improve the treatment. Melanin pigment is found at high concentrations in ocular tissues, and many clinical drugs bind to it. Targeting the pigment to obtain sustained drug action in ocular tissues could be a feasible approach for improving ocular drug therapy. Therefore, this thesis project aimed to study the effects of pigment binding on pharmacokinetics in the eye with in vitro, in vivo and in silico methods. In addition, we investigated drug binding to the vitreous humor, as it may participate in modulating ocular pharmacokinetics and has been studied only scarcely. Melanin is a negatively charged, hydrophobic polymer, and it is expected to bind all basic and lipophilic drugs to some extent. Melanin is located in intracellular melanosomes, which are cell organelles surrounded by a lipid membrane, and are expected to have an acidic intraluminal pH. Due to the location of melanin, cellular and physiological factors act in concert with the binding to the melanin polymer, to impact ocular pharmacokinetics of melanin-binding drugs. We investigated pigment binding and related cellular and physiological factors with seven small molecule drug or drug-like compounds: chloroquine, timolol, nadolol, propranolol, methotrexate, 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) and diclofenac. A pH-dependent binding (pH 5.0 vs. 7.4) to isolated porcine ocular melanin was observed, mainly with the acidic compounds that are less negatively charged and bind better at pH 5.0. Therefore, pH plays an important role in drug-melanin binding in the case of acidic drugs, but is less important in the case of basic drugs. The binding parameters, maximum binding capacity and affinity, were reliably calculated with the Sips binding isotherm instead of the commonly used Langmuir isotherms. The Sips isotherm is in line with the heterogeneous nature of the melanin surface to where the drugs bind, and, therefore, is better suitable for parameter analysis. Cellular uptake and intracellular binding in pigmented retinal pigment epithelial cells were shown to correlate with melanin binding, but other factors, such as lipophilicity of the drug, also need to be taken into account. Pharmacokinetic simulations of the retention of drugs in pigmented posterior segment tissues demonstrated that low drug permeability in the plasma and melanosomal membranes and the entrapment of positively charged drugs in the melanosome increases melanin binding related retention in these tissues. In addition, both the intracellular binding experiments with pigmented cells and the pharmacokinetic simulations showed that only a small fraction (~0.01%) of the highest binding drug of this study, chloroquine, is in the free form inside the cells. The free form of the drug elicits the drug action (beneficial or harmful), and it is, therefore, important to differentiate between the free and total drug inside the cells when assessing drug response. Furthermore, we demonstrated the distribution and retention of 123I-chloroquine in the eyes of pigmented but not albino rats after intravenous administration, establishing the use of single photon emission computed tomography/computed tomography imaging in monitoring melanin binding related kinetics in vivo. All in all, in the variety of experiments performed, melanin binding was shown to have a major impact on ocular pharmacokinetics in pigmented tissues. In addition to investigating melanin binding, drug binding to the vitreous humor was studied with a cassette of 35 clinical small molecule drugs. The binding was rather low and vitreal binding was concluded to have only a modest effect on ocular pharmacokinetics. In conclusion, this thesis project generated important information of the extensive pharmacokinetic impact of drug-melanin binding in ocular tissues. In addition, we demonstrated the moderate pharmacokinetic impact of vitreal drug binding, which is not comparable to melanin binding. The computational models developed on melanin binding and its pharmacokinetic implications can be used in drug discovery and development. The in vitro methods can also be implemented to the industry scale drug development process. The results obtained support the feasibility of using melanin targeting to attain sustained action in pigmented ocular tissues, but more research into the approach is needed before it can be employed in practice.
  • Lipiäinen, Tiina (Helsingin yliopisto, 2018)
    The solid-state form can directly affect the quality of a pharmaceutical product and solid-state transformations can lead to altered therapeutic effects. Formulation and manufacturing processes may impact the solid-state form and stability. There is a need to detect, quantify and control the solid-state behaviour during processes and storage. Reliable analytical methods are crucial. The overall aim of this thesis was to evaluate strategies for controlling stability and analysing solid-state forms in pharmaceutical powders. New excipients for stabilisation of spray-dried protein formulations, as well as Raman spectroscopy-based methods for solid-state quantification, new in the pharmaceutical field, were investigated. Melibiose and isomalt were studied as potential new stabilising excipients for spray-dried protein formulations. The process behaviour of these two carbohydrates, as well as the physical stability of their amorphous powders, was evaluated in comparison to sucrose and trehalose. Both could be spray dried into amorphous powders, but melibiose was more suitable for spray drying processes and showed better physical stability than isomalt. The protein-stabilising efficacy and process behaviour of melibiose in spray-dried protein formulations was compared to the current standard excipient, trehalose. Protein formulations with melibiose could be spray dried into amorphous powders that were physically stable, contained lower moisture contents and protected protein activity at least as well as formulations with trehalose. Low-frequency Raman spectroscopy for quantitative analysis of solid-state form mixtures was investigated in order to evaluate its potential advantage over established mid-frequency Raman spectroscopy. Low-frequency Raman spectroscopy was found better because of higher signal intensity and solid-state sensitivity. Time-gated Raman spectroscopy was tested for quantitative solid-state analysis of fluorescent pharmaceutical powder mixtures. Fluorescence interference sometimes limits the feasibility of Raman spectroscopy. Both standard multivariate analysis and kernel-based methods were used for data analysis. It was found that time-gated Raman spectroscopy, particularly when combined with kernel-based data analysis methods, provided benefits for the quantitative analysis of materials suffering from fluorescence. In summary, melibiose was identified as a promising excipient to stabilise spray-dried protein formulations. Low-frequency and time-gated Raman spectroscopy were found advantageous for solid-state analysis, also with fluorescent materials, and they may be useful techniques for various solid-state monitoring applications.
  • Almeida, Patrick (Helsingin yliopisto, 2018)
    Recent breakthroughs in nanotechnology have paved the way for a new era in cancer medicine. Among the myriad of nanotechnology-based systems that have been revolutionizing the field of cancer nanomedicine, porous silicon (PSi) nanoparticles have recently emerged as a promising nanoplatform, owing to advantageous physicochemical and biological properties. Nevertheless, the successful establishment of PSi nanoparticulate systems as effective cancer nanomedicines is challenged by several shortcomings associated with the instability in biological fluids, the poor tumour targeting efficiency and unfavourable pharmacokinetics, the limited capacity to overcome extra and intracellular biological barriers, and the ubiquitous and uncontrolled release of the therapeutic payloads. This dissertation aimed at designing and developing novel strategies, including the surface modification of PSi nanoparticles with biofunctional polymers and the engineering of advanced multifunctional PSi-based nanocomposites, in order to overcome some of the aforementioned deadlocks, improving the tumour targeting and drug delivery efficiencies, and ultimately potentiating the application of PSi nanomaterials in cancer nanomedicine. First, the biofunctionalization of PSi nanoparticles with a hyaluronic acid (HA) derivative was proven to improve the colloidal and plasma stabilities and to significantly enhance the cellular internalization of the nanosystems in breast cancer cells. The HA-modified PSi nanoplatforms exhibited higher affinity and endocytic activity in the cells overexpressing the CD44 receptor, thus evidencing a great potential for further development as active targeted drug delivery systems to CD44-overexpressing tumours. Next, a bilayered zwitterionic PSi nanocomposite was fabricated by successive conjugation of polyethyleneimine and poly(methyl vinyl ether-alt-maleic acid) polymers on the surface of PSi nanoparticles. In addition to satisfactory cytocompatibility, and high colloidal and plasma stabilities, the designed polymeric surface modification was shown to enhance the non-specific cellular association and uptake, and to improve the intracellular trafficking of the PSi nanoparticles in breast cancer cells. Moreover, this strategy contributed to increase the drug loading of methotrexate (MTX), sustain the release of the drug and potentiate the in vitro antiproliferative effect of the MTX-loaded PSi nanocarriers. In addition, PSi nanoplatforms were used to engineer multifunctional PSi-based nanocomposites, envisioned for cancer therapeutic and theranostic applications. In one approach, both sorafenib-loaded PSi and gold nanoparticles were simultaneously encapsulated into a self-assembling polymeric nanocomplex. In another study, the same nanocomplexes were used to encapsulate DNA-capped PSi nanoparticles, as an innovative strategy to bioresponsively deliver hydrophilic and hydrophobic drug molecules into the cytosolic compartment of cancer cells. The potential of the fabricated multifunctional PSi-based nanocomposites stemed from the versatility to incorporate a combination of nanosystems, hydrophilic or hydrophobic drug molecules, and fluorescent dyes within a single nanostructure, and the capability to enhance the cellular interactions, endocytosis and cytoplasmic delivery of the encapsulated nanoparticles and therapeutics. In conclusion, the developed PSi-based nanocomposites exhibited great potential for cancer targeting and drug delivery, representing an advanced contribution for the successful implementation of PSi nanomaterials as the next generation of cancer nanomedicines.
  • Saarinen, Jukka (Helsingin yliopisto, 2018)
    In the pharmaceutical industry, novel analytical techniques are required to gain important insights about new drug candidates and their formulations as early as possible. This information can be used to develop more efficient, safe and also economically profitable medicines. Microscopy techniques can be used for example to follow the fate of nanoparticles in cells and tissues, but also to monitor the changes in solid-state forms of drug molecules during drug development and storage. The overall aim of the Thesis was to evaluate the capability of non-linear optical imaging, especially coherent anti-Stokes Raman scattering (CARS), second harmonic generation and sum-frequency generation (SHG and SFG) microscopies, in pharmaceutical applications including imaging of live cells, nanoparticle cellular uptake and pharmaceutical solid-state analysis. First, the capability of CARS microscopy to image live cell cultures on pharmaceutically relevant membrane inserts was evaluated. It was found that, label-free CARS microscopy can be used to image Caco-2 cells (used in drug permeation studies) grown on PTFE inserts in a non-destructive manner. Label-free CARS microscopy could probe lipid droplets and the size of lipid droplets increased substantially over a 21- day culturing period, which is important in the context of drug permeation studies, since lipid content of the cells will influence drug permeation. Next, CARS microscopy was successfully used to probe non-fluorescent nanocrystals in cells in a label-free and chemically-specific manner. In addition, electron microscopy could be used to visualize the subcellular location of nanocrystals in cells with nanometer spatial resolution in a chemically-specific manner by using a developed correlative imaging method with CARS and EM. At the end, multimodal CARS and SHG/SFG imaging was used to visualize the distribution and crystallization of indomethacin and its solid states on tablet surfaces. The combined use of two imaging modalities is beneficial, since data provided by using two techniques relying on different mechanisms (detection of molecular vibrations (CARS) and SFG signal produced by non-centrosymmetric crystals), can support each other. In summary, it was demonstrated that non-linear optical imaging can be a very useful tool in pharmaceutical applications including imaging of live cells, nanoparticle cellular uptake and solid-state analysis. The results were obtained by using a commercially available microscope, which suggests that there is plenty of potential in these techniques to be applied on a wider scale. The use of these techniques is likely to increase with further instrument commercialization in the near future.
  • Ferreira, Mónica (Helsingin yliopisto, 2017)
    Currently, there is no major discovery of an effective cure to restore the function of an injured heart, despite the existing and developing therapies. While existing options ameliorate the care of myocardial infarction (MI) and heart failure patients, cardiac stem cell therapy has only recently shown positive results in clinical trials, and thus there is an urgent medical need to develop advanced therapeutic entities to reverse this disease burden. The employment of biomaterials as potential therapeutics for MI is at the pre-clinical stage. Particulate systems are arising as a promising tool to provide minimally invasive treatment, an important aspect to take into account for clinical translation and patient compliance. Porous silicon (PSi) and spermine-acetalated dextran (AcDXSp) are emerging biomaterials for applications in varying biomedical fields. Drug delivery is one of these fields benefiting from the materials’ properties, such as biocompatibility, biodegradability, customized particle preparation, surface functionalization, simple yet efficient drug loading, and tunable release of the therapeutic cargos. Therefore, the aim of this thesis was to develop multifunctional PSi and AcDXSp platforms for targeted drug delivery to and imaging of the ischemic heart. Initially, the biocompatibility of PSi-based carriers of different sizes and surface chemistries was evaluated. Secondly, three different PSi-based nanosystems were developed, functionalized with a metal chelator for radiolabeling and three different peptides (atrial natriuretic peptide (ANP) and two other heart-homing peptides), with the aim to screen the targetability of the nanoparticles to the ischemic heart. All the nanosystems showed no toxicity up to 50 µg/mL concentration, and cell–nanoparticle interaction studies in cardiomyocytes and non-myocytes revealed a preferential cellular interaction with ANP-functionalized nanoparticles in both the cell types, through the natriuretic peptide receptors (NPRs) present at the cell surface. Thirdly, the ANP-PSi functionalized nanoparticles were PEGylated in order to improve the colloidal stability and enhance the circulation time. Upon labeling with radioisotope Indium-111, the ANP-PSi nanoparticles displayed a preferential accumulation and selectivity towards the endocardial layer of the ischemic heart. In vivo delivery of a cardioprotective small drug molecule from the ANP-PSi showed attenuation of the extracellular signal-regulated kinase pathway that is involved in the hypertrophic signaling of the injured heart. Lastly, and in parallel, the development of functionalized and dual-loaded AcDXSp nanoparticles for potential application in cellular reprogramming was proven successful, by utilizing acidic pH-triggered drug delivery of the two poorly water-soluble cargos. The incubation of non-myocytes with ANP-functionalized AcDXSp nanoparticles showed therapeutic modulation of key signaling pathways involved in the direct fibroblast reprogramming into cardiomyocytes. Overall, PSi and AcDXSp-based (nano)particulate systems were developed, bringing new insights about potential therapeutic advances in the applicability of imaging and targeted delivery of relevant pharmacological molecules to the ischemic heart with a minimally invasive therapeutic approach.
  • Räsänen, Riikka-Marjaana (Unigrafia, 2017)
    This thesis describes the applicability of different types of IMS instruments in the direct measurements of gaseous and solid samples and in fundamental studies of gas-phase ion chemistry. A handheld chemical detector containing an aspiration ion mobility spectrometry (AIMS) was applied in the monitoring of gas phase explosive triacetone triperoxide (TATP) from air flow. The instrument-normalized detection threshold (20 pA) was exceeded already with the lowest sample concentration of 0.3 mg m−3. The response time of the instrument was less than five seconds. AIMS was also used to monitor chemical changes in the headspaces of the chambers containing microbe contaminated and sterile particle board samples in humid conditions. It was possible to separate the distinct chemical profiles of the chambers with sterile and microbe-contaminated specimen by principal component analysis. Overall, AIMS was found to be an adequate technique in dynamic screening of TATP and in monitoring of the changes in the microbe metabolism. Ambient ionization techniques, direct analysis in real time (DART) and desorption atmospheric pressure photoionization (DAPPI), were combined with travelling wave ion mobility-mass spectrometry. In the surface analysis of almond, pharmaceuticals, vitamin tablets and dried blood spot sample, the ion mobility separation reduced the chemical noise in the mass spectra and increased the signal-to-noise ratio. In the comparative studies of DAPPI and DART ionization, the limits of detection were between 30−290 and 330−8200 fmol for DAPPI and DART, respectively, for the tested compounds bisphenol A, benzo[a]pyrene, ranitidine, cortisol, and α-tocopherol. Finally, the reactions of phenol and fluorinated phenols with Cl− in ambient pressure were investigated by drift tube ion mobility-mass spectrometry. For the least fluorinated phenols (phenol, 2-fluorophenol and 2,4-difluorophenol) with the lowest gas-phase acidities, the Cl− adducts [M+Cl]− and [2M+Cl]− were the major products in both low and high sample concentration. For the highly fluorinated phenols (2,3,6-trifluorophenol and pentafluorophenol), [M−H]− and [2M−H]− were the main products in high sample concentration. In low concentration [M−H]− and [M+Cl]− were the main products. In case of pentafluorophenol (PFP), in high temperature conditions the dimer was [2PFP−H]− instead of [2PFP+Cl]−. In conclusion, IMS has many advantages and application possibilities. It allows the rapid detection and continuous monitoring of volatiles directly from ambient air. IMS can also be used as a pre-separation technique in ambient mass spectrometry, without increasing the total analysis time remarkably. In IMS, it is also possible to study gas phase reactions in ambient conditions. Some of the IMS applications presented in this thesis could be developed further to be a permanent part of routine monitoring, analysis, and research work. For example, in the fundamental studies of phenols, the possibility to use updated versions of the instruments could improve the accuracy of the experiments. In addition, broader studies with several experimental conditions, would increase the possibility to develop the method further, especially in the monitoring of TATP, and building material and microbe emissions from the gas phase with AIMS.
  • Laine, Niina (Helsingin yliopisto, 2017)
    BACKGROUND AND OBJECTIVES Rational use of antimicrobials is paramount due to increasing bacterial resistance and a lack of novel antimicrobials. Investigating the clinical use and consumption of antimicrobials aids in the prudent use of these drugs in a tertiary paediatric hospital. The purpose of this study was to obtain detailed information on the use of antimicrobials in a tertiary Children’s Hospital, Helsinki University Hospital, in order to support prudent, safe and efficient use of antimicrobials. The objectives were the following: 1) To evaluate the appropriateness of antimicrobial therapy (AMT) in children with blood culture positive infections (Study I), 2) To investigate the consumption of antimicrobials in the hospital in Defined Daily Doses (Study II), 3) To record the prevalence of off-label use of antimicrobials in neonates (Study III) and last, 4) To analyse the occurrence of antimicrobial medication errors in children (Study IV). MATERIALS AND METHODS The Children’s Hospital, University of Helsinki, is a tertiary hospital in Finland. In Study I, data on 149 children (0–17 years) with blood culture positive hospital infections between 2005 and 2012 were collected. In Study II, the consumption of antimicrobials in Defined Daily Doses (DDDs according to the Anatomical Therapeutic Chemical (ATC)/DDD index) was investigated retrospectively between 2003 and 2013. In Study III, the prevalence of off-label use of antimicrobials was investigated in three different paediatric cohorts. The largest cohort consisted of premature NICU patients (450–2000g) with blood culture positive infections and antimicrobial therapy given between 2005 and 2014 (N=282). In Study IV, the types of documented antimicrobial errors were analysed. The errors were reported by healthcare professionals using a voluntary web-based error reporting system, HaiPro between 2009-2014. Overall, different types of methods were used regarding quantitative and qualitative analysis and retrospective reviews of electronic patient records and registry data. RESULTS The AMT was inappropriate in 17% (26/149) of patients with blood culture positive infections (Study I). Three of these patients received antimicrobials that were totally ineffective according to in vitro data. During 2003 and 2013, the use of many beta-lactam antimicrobials increased. The most notable change was in the use of carbapenems, which increased by 110% during the study period (Study II). A total of 18% (51/282) of premature neonates with blood culture positive infection received at least one off-label antimicrobial (Study III). An increase in birth weight was found to statistically significantly decrease the probability of off-label usage (odds ratio=0.85 for 100g increase in birth weight, p-value < 0.001). In Study IV, there were 157 antimicrobial errors reported in 149 patients from four wards (GEN, NICU, HEM-ONC and INF). Two errors were reported as clinically significant (2/149, 1%). Most of the errors occurred with drugs with high consumption, such as cefuroxime (15/157, 10%) and penicillin G (15/157, 10%). CONCLUSIONS These studies gave a useful overall picture regarding AMT and the use of antimicrobials at the Children’s Hospital. More attention should be paid to appropriate AMT, and training of prescribers should be provided. This thesis provides a window into issues that undermine the quality of care regarding hospital infections in paediatrics and aids the launch of an antimicrobial stewardship program (ASP) in the Children’s Hospital.
  • Tseng, Kuan-Yin (UNIVERSITY OF HELSINKI, 2017)
    Stroke is one of the leading causes of death and a major cause of disabilities in adults. More than half of stroke victims suffer some type of disability, ranging from different levels of minor weak- ness in a limb to a complete loss of mobility. Currently, treatment of stroke requires a stringent re- habilitation programs. Nevertheless, two thirds of all patients will still have some type of difficulty with regular daily activities. Recent experimental findings raise the possibility that functional improvement after stroke may be achieved through neuronal replacement by endogenous neural stem cells (NSCs) residing in the adult brain. Therefore, additional understanding of the properties of NSCs will help to identify their optimal potential in cell-based therapy. Neurotrophic factors are a family of proteins that are important in neuronal development and function, and have been studied as possible drugs for ischemic brain injury. In addition to Brain-Derived Neurotrophic Factor (BDNF) and Glial cell line-Derived Neurotrophic Factor (GDNF), Mescenphalic Astrocyte-Derived Neurotrophic Factor (MANF) and Cerebral Dopamine Neurotrophic Factor (CDNF), that form a distinct family of evolutionary conserved proteins with neuroprotective effects, have potential in the treatment of stroke. While MANF has been shown to protect cortical neurons from death in a rodent model of ischemic brain injury, the effects of post-stroke MANF ad- ministration on cellular processes during the recovery phase are poorly understood. To shed light on the possible regenerative potential of MANF for the injured brain, we need to first investigate the roles of endogenous MANF in neural stem cells (NSC) in a normal or pathological condition. We developed and optimized a work platform for studying the regulation and effect of MANF on biological properties of NSCs and cortical development. Our findings reveal an important role of MANF in neurite outgrowth and neuronal migration in the developing cortex. In addition, we demonstrated that endogenous MANF has the potential to protect NSCs against oxygen and glucose-deprivation conditions. Next, using neurosphere and subventricular zone (SVZ) explant cultures, we further studied the effect of MANF administration on cell differentiation and migration. We presented the data that exogenously added MANF can induce neural/glial differentiation and promote cell migration out of SVZ explants. Also, utilizing the advantage of NSCs as a target for MANF, we discovered that exogenous MANF can induce the phosphorylation of STAT3 in NSCs. Finally, we used the rat model of ischemic stroke to compare the effects of MANF and GDNF in neurogenesis after stroke. While injection of GDNF into lateral ventricle has a strong mitogenic effect to increase neurogenesis in SVZ, it does not induce migration of neuroblasts towards the ischemic area. In contrast, MANF facilitates the migration of neuroblasts towards the lesioned cortex. Regarding long-term infusions in the peri-infarct zone, both GDNF and MANF recruited the neuroblasts in the infarct area. However, only MANF accelerated functional recovery after stroke. In summary, this work has extended the knowledge of MANF’s capacity for neuronal differentiation as well as migration, and the regenerative capacity for its therapeutic use in further studies.
  • Lantto, Tiina A. (Helsingin yliopisto, 2017)
    Plant phenolics and extracts are able to affect cell signalling associated with regulated cell-death mechanisms. Such mechanisms play a crucial role in the normal homeostasis of an organism, but inadequately functioning cell-death machinery is a component of the development of complex diseases, such as cancer, where cells divide in an uncontrolled manner. Apoptosis is the most studied regulated cell-death mechanism associated with cancer. One of the key triggers of and contributors to apoptotic cell death is a tumour suppressor p53. This protein is constantly produced, though it is activated only by several cellular stress responses, such as endoplasmic reticulum stress. The purpose of this study is to investigate the cytotoxic and apoptotic properties of three plant phenolics – curcumin, resveratrol and quercetin – and seven plant extracts – basil, juniper, laurel, lemon balm, parsley, and Siberian pine – in cancerous neuroblastoma and melanoma, and non-cancerous fibroblast cell models. The emphasis of the work is on plant extracts due to their claimed additive or synergistic effects on cellular mechanisms. The effects of different treatments are determined by two cell-viability tests, followed by Western blot assays of the amounts of p53, anti-apoptotic Bcl-2, and inflammatory p65. Apoptotic events are defined by the activity of caspase 3 and DNA fragmentation. Further testing to reveal a broader spectrum of effects is defined by the cDNA RDA method in order to investigate genes expressed differently in treated and in untreated cells. The results of this study support existing knowledge of the effects of single-plant phenolics, and reveal new mechanisms for the activity of plant extracts. Possible synergistic or additive effects of juniper plant extract on apoptosis through endoplasmic reticulum stress are observed. Plant phenolics and extracts may provide a unique pool of drug candidates for the prevention or treatment of cancer. The use of plant extracts as drug candidates is especially interesting due to the possible synergistic or additive effects they achieve at low concentrations.
  • Pessi, Jenni (Helsingin yliopisto, 2017)
    This thesis consists of two parts, particle formation and analysis. In the first part, particle formation in microfluidic devices and in devices employing supercritical fluids is investigated, and in the second part, essential issues in analytical methods for determining drug release and solid-state properties are addressed. Microfluidic technology was employed to produce microcapsules for protein formulations. The microcapsules were produced with a biphasic flow to create water-oil-water double emulsion droplets with ultrathin shells. All the particles were found to be intact and with a particle size of 23 - 47 µm. The encapsulation efficiency of bovine serum albumin in the microcapsules was 84%. This study demonstrates that microfluidics is a powerful technique for engineering formulations for therapeutic proteins. A new, robust, stable, and reproducible method based on expansion of supercritical solutions using carbon dioxide as a solvent was developed to produce nanoparticles. The method, Controlled Expansion of Supercritical Solution (CESS), uses controlled mass transfer, flow, pressure reduction, and particle collection in dry ice. CESS offers control over the crystallization process as the pressure in the system is reduced according to a specific profile. Controlled pressure reduction keeps the particle growth and production process stable. With CESS, we produced piroxicam nanoparticles, 60 mg/h, featuring narrow size distribution (176 ± 53 nm). The Lyophilic Matrix (LM) method was developed for investigating dissolution rates of nanoparticles, powders, and particulate systems. The LM method is based on its ability to discriminate between non-dissolved particles and the dissolved species. In the LM method, the test substance is embedded in a thin lyophilic core-shell matrix. This permits rapid contact with the dissolution medium while inhibiting dispersion of non-dissolved particles without presenting a substantial diffusion barrier. By minimizing method-induced effects on the dissolution profile of nanopowders, the LM method overcomes shortcomings associated with current dissolution tests. Time-gated Raman spectroscopy was applied for solid-state analysis of fluorescent powder mixtures. A setup with a 128 × (2) × 4 CMOS SPAD detector was used for the quantitative analysis of solid-state forms of piroxicam. Time-gating provides an instrumental method for rejecting the fluorescence signal. This study demonstrated that traditional PLS analysis of time-gated Raman spectra resulted in mean RMSE of 4.1%. The time-gated Raman spectroscopy method shows potential for relatively routine quantitative solid-state analysis of photoluminescent pharmaceuticals.
  • Ruokolainen, Miina (Helsingin yliopisto, 2017)
    Redox reactions play an important role in human physiology and pathophysiology. For example, oxidative stress and free radical-mediated oxidation of proteins and lipids are implicated in several diseases such as Alzheimer’s and Parkinson’s disease. Oxidation reactions belong also to the most important phase I metabolism pathways of drugs, which can give rise to pharmacologically active or toxic metabolites. The established methods for in vitro drug metabolism studies, e.g. methods using hepatocytes, human liver microsomes (HLMs), and recombinant enzymes, are relatively time-consuming and expensive. Thus, the potential of several nonenzymatic oxidation methods, such as those based on metalloporphyrins, electrochemistry (EC), and Fenton reaction, have been explored for metabolism studies. However, new methods need to be developed to enable rapid production of drug metabolite standards and since none of the above nonenzymatic methods allow comprehensive prediction of phase I drug metabolism. The titanium dioxide (TiO2) photocatalysis method was developed and applied to evaluate the effect of phosphorylation of tyrosine on the oxidation of (phospho)peptides with the same sequence but different phosphorylation states. The results obtained using ultra-high-performance liquid chromatography – mass spectrometry (UHPLC-MS) show that nonphosphorylated tyrosine was the amino acid most susceptible to hydroxyl radical-initiated oxidation, but oxidation of tyrosine was in most cases inhibited by its phosphorylation. The feasibility of TiO2 photocatalysis for imitation of in vitro phase I HLM metabolism of small drug molecules was studied using UHPLC-MS and compared with the electrochemically assisted Fenton reaction (EC-Fenton) and EC. TiO2 photocatalysis, EC-Fenton, and EC imitated 44%, 31%, and 11%, respectively, of the in vitro phase I HLM metabolites of four model compounds. As TiO2 photocatalysis proved most feasible for the imitation of in vitro phase I HLM metabolism, its feasibility for imitation of in vitro phase I HLM metabolism of five anabolic steroids was also examined. TiO2 photocatalysis was able to imitate over half of the hydroxylation and dehydrogenation metabolites, but its imitation of the metabolites resulting from combinations of these reactions was considerably poorer. To enable even more rapid experiments to study biologically relevant oxidation reactions, TiO2-photocatalysis was simply integrated with desorption electrospray ionization (DESI)-MS by using the same TiO2-coated glass wafer for photocatalytic reactions and DESI-MS analysis. This new method enabled high-throughput investigation of photocatalytic oxidation reactions, as demonstrated using 12 model compounds, and imitation of several drug metabolism reactions of three model compounds studied in more detail. In conclusion, TiO2 photocatalysis proved a feasible method for oxidation of compounds with different polarities. TiO2 photocatalysis cannot predict drug metabolism comprehensively, but offers a potential method for rapid, simple, and inexpensive study of oxidation reactions of biomolecules and imitation of several drug metabolism reactions. Preparative scale synthesis of oxidation products by TiO2 photocatalysis is likely an alternative application of the method, but this remains to be demonstrated.