Faculty of Pharmacy


Recent Submissions

  • Kilpeläinen, Tommi (Helsingin yliopisto, 2021)
    Most neurodegenerative diseases (NDD) are characterized by accumulation of toxic protein species leading to the loss of movement and cognitive disorders. In the two most common NDD Parkinson´s disease (PD) and Alzheimer´s disease (AD), there are almost 60 million patients worldwide. In the U.S, there were one million PD patients and an estimated economic burden of 51.9 billion dollars in 2017. As there is no disease-modifying therapy available, NDD lead to loss of life quality and eventually to untimely death. Current drug therapies for PD can only relieve symptoms of the disease by increasing dopaminergic activity in the brain but they do not address the underlying mechanisms of the disease such as clearance of detrimental protein aggregates. In previous studies, prolyl oligopeptidase (PREP) has been shown to negatively regulate autophagy and enhance the aggregation of alpha-synuclein (αSyn). Thus, PREP inhibitors have been shown to reduce aggregation and increase clearance of αSyn aggregates via enhanced autophagy. These mechanisms are mediated by protein-protein -interactions (PPI) of PREP with other proteins. Several small molecular inhibitors of PREP have been developed to target memory disorders, cancer, and NDD. However, none of these PREP inhibitors are in therapeutical use. The aim of this study was to investigate the effect of these inhibitors in two neurodegeneration-related functions of PREP: autophagy and αSyn dimerization. A further aim was to synthesize novel PREP ligands targeted to modulate these PPI-mediated functions of PREP. In the first part of this study, a structurally diverse set of 12 potent PREP inhibitors were tested in in vitro assays of autophagy and αSyn dimerization. The study revealed that the least potent inhibitor with IC50-value of 1010 nM had the most prominent effect on reduction of αSyn dimers and enhancing autophagy, while some of the highly potent inhibitors with IC50-value ranging from 0.32 to 1.2 nM did not have an effect on autophagy or αSyn dimerization. In the second part of the study, novel 4-phenylbutanoyl-α-aminoacyl-2(S)-tetrazolyl-pyrrolidines were designed and synthesized to target αSyn dimerization. Synthesized compounds had IC50-values ranging from 12 nM to 200 000 nM, but they all reduced αSyn dimerization in a cellular assay. Furthermore, molecular docking studies showed that these novel compounds have another putative binding mode to PREP compared to typical PREP inhibitors. These findings combined with the findings from the first part of the study suggest that the PREP mediated reducing effect on αSyn dimerization and enhancing effect on autophagy is not dependent on the magnitude of PREP inhibition but more likely due to conformational stabilization in PREP caused by ligand binding. Another speculative explanation is that PREP has another binding site, which modulates the conformation of PREP and thereby the PPI mediated functions of PREP. In the third part of the study, a human αSyn transgenic mouse, carrying A30P and A53T point mutations in αSyn was characterized and evaluated as a model for early onset PD. The model displayed behavioral alternations at an early age with differences in dopaminergic neurotransmission in the nigrostriatal pathway and accumulation of oligomeric αSyn leading to decreased tyrosine hydroxylase in the striatum and substantia nigra. Furthermore, we evaluated the effect of seven days treatment by the novel PREP inhibitor, HUP-55, in this model. Results in this study showed that HUP-55 was able to decrease the level of oligomeric αSyn in the striatum and substantia nigra. In summary, this study revealed and highlighted the disconnected structure-activity relationships between inhibition of the proteolytic activity and the PPI mediated functions of PREP. Moreover, this finding led to the development of a novel PREP ligand that was successfully tested in a PD mouse model.  
  • Siiskonen, Satu Johanna (Helsingin yliopisto, 2021)
    This doctoral dissertation summarises current knowledge on risk factors for the most common types of skin cancer and describes some of the important methodological challenges related to research on skin cancer aetiology. The three original studies each assess a different type of risk factor for cutaneous squamous cell carcinoma (SCC) or cutaneous malignant melanoma (CM) using large data sources in the USA and/or the Netherlands. Study I was one of the first published genome-wide association studies on SCC. The most significant finding was the association between a single nucleotide polymorphism (SNP) rs8063761 in the DEF8 gene and SCC, which has since been replicated by other research groups. The role of the DEF8 gene in skin carcinogenesis is yet to be determined. A single finding from a genome-wide association study with a modest effect size has no clinical value in screening individuals at high risk for SCC. It can, however, be useful when constructing polygenic risk scores to predict an individual’s genetic predisposition to SCC development. Study II was a large cohort study with time-dependent variables to investigate the association between alcohol intake and SCC. Alcohol consumption was associated with SCC and SCC in situ in a dose-dependent manner in men and women. The results of this study also suggest an association between white wine consumption and SCC, when the analyses were adjusted for the total alcohol intake. However, residual confounding by other factors, such as time spent in the sun, cannot be ruled out. Alcohol use is a modifiable risk factor for many cancers and other diseases, and its use should be minimised. Study III reports an association between the use of phototoxic quinolone antibiotics and propionic acid derivative analgesics, and CM. Even short-term use may be relevant, and the risk estimates were different for men and women. It cannot, however, be excluded that the detected associations are partly due to bias or confounding by unmeasured factors. Although the relationship between phototoxic reactions and development of CM is still a topic of debate, phototoxic reactions themselves can cause significant morbidity. Healthcare professionals should therefore advise patients to avoid sun exposure while taking phototoxic drugs. Identifying and assessing risk factors for skin cancer is methodologically challenging due to lacking knowledge on relevant exposure time periods and often incomplete or absent information on exposure to UV radiation and other known strong predictors of skin cancer, such as skin phenotype and the use of immunosuppressants. The data source used in etiologic skin cancer research should, ideally, include information on the known risk factors and validated skin cancer diagnosis and allow for careful examination of relevant exposure time periods.  
  • Štukelj, Jernej (Helsingin yliopisto, 2021)
    The two parameters used to characterize the effect of a drug in the body are potency and selectivity. Despite their importance, the biopharmaceutical classification system (BCS) and the developability classification system (DCS) are constructed based on permeability and solubility. Furthermore, solubility is not only a molecular property as it is highly affected by the solid-state of a compound. Thus, obtaining a complete understanding of the solid-state impact on the solubility of a drug candidate early in the drug development process is crucial for well-informed decision-making. The overall aim of this thesis was to detect, investigate and quantify the impact of different solid-state forms on the solubility of pharmaceutical compounds using the image-based single-particle analysis (SPA) method. First, the apparent solubility of polymorphs in different solvents was measured. It was demonstrated that the solubility ratio between the two polymorphs is determined by the difference in Gibbs free energy between the polymorphs and it is not affected by changes in pH, ionic-strength and surfactant concentration in dissolution media. Next, the amorphous solubility of a particulate quench-cooled amorphous sample was measured directly. The results correlated well with the two orthogonal methods – standardized supersaturation and precipitation method, and theoretical estimation based on thermal analysis. Moreover, the direct approach of measuring amorphous solubility has proven superior when dealing with highly hygroscopic and fast crystallizing samples. Furthermore, the newly established direct approach of measuring amorphous solubility was then, concurrently with X-ray powder diffraction, used to track two amorphous samples stored at different conditions over an extended period of time. Differences in the crystallization process and its impact on solubility were observed. Machine-vision-detected solid-state changes were demonstrated to provide additional information for the interpretation of the measured pH-solubility profiles of pharmaceutical salts. Based on pH-solubility profiles, values such as pKa, pHmax and Ksp were easily obtained. Moreover, the potential effect of the microenvironmental pH shift was evaluated in the context of solubility measurements. Finally, the solubility of extremely poorly soluble drug – itraconazole – and its co-crystals was measured across a wide pH range and also in biorelevant buffers. A considerable improvement in solubility was detected upon co-crystal formation. Moreover, the solubility of itraconazole in biorelevant buffers was in good agreement with the results in literature obtained previously by the conventional shake-flask method. In summary, all of the above was achieved with less than 100 μg of a sample per measurement. Moreover, non-specificity and the demonstrated broad applicability make the SPA method highly suitable for use in the early stages of the drug discovery and development. The possibility of acquiring a broad level of physicochemical characterization using a single analytical method could significantly accelerate and improve data acquisition leading to enhanced understating of solid-state impact on solubility. Furthermore, with the data quality comparable to the current ‘gold standard’ technique the SPA method could thread the path towards more accurate in silico solubility predictions. Thus, eventually leading to more informed decision-making processes and, finally, optimized and more affordable drug products.
  • Karhu, Tuuli (Helsingin yliopisto, 2021)
    Heart failure is a common costly disease that remains one of the leading causes of mortality in the world. Current treatments benefit heart failure patients by relieving the symptoms and improving the quality of life; however, they do not provide a cure for the disease. The adult human heart has a very limited capacity to regenerate cardiomyocytes. Therefore, following cardiac injury, the cell loss results in the formation of a fibrotic scar and consequent remodelling process, which includes thickening and stiffening of the ventricular wall. Eventually, this leads to impaired cardiac function. The fundamental goal of heart failure therapy is to restore the cardiac function and to prevent the pathological remodelling process. The transcription factor GATA4 plays an important role in cell survival, myocardial regeneration, and cardiomyocyte hypertrophy, and is therefore considered as an attractive drug target for cardiac repair. On the other hand, several studies have suggested that protein kinase C (PKC) plays a role in cardiac fibrosis. However, the results are conflicting and the role of PKC specifically in cardiac fibroblasts is unclear. Early drug discovery projects have traditionally used immortalized cell lines and primary cells from experimental animals to assess the effects of novel compounds. The translatability of the results from these models to human, however, is limited. Today, the emergence of the human induced pluripotent stem cell (hiPSC) technology allows for the production of human-based cell models, which more closely resemble the physiological cell types. The aim of this thesis project was to characterise pharmacological and toxicological effects of small molecules targeted to GATA4 or PKC, to compare different cell models in in vitro compound testing, and to establish a chronic cardiotoxicity model using human-based cardiomyocytes. In the study, nine different cell models were utilised, including hiPSCs, cardiomyocytes derived from hiPSCs, cardiomyocytes isolated from neonatal rats, and cardiac fibroblasts isolated from adult mice. The effects of the small molecules on cell viability, proliferation, and morphology were investigated using colorimetric cytotoxicity assays, immunocytochemistry, and high-content analysis (HCA). The effects on gene expression, protein levels, and cellular kinase activity were studied using the quantitative polymerase chain reaction (qPCR), western blotting, and AlphaLISA® technology, respectively. The toxicity screening of GATA4-targeted compounds revealed a characteristic molecular structure that is predominantly responsible for the toxic outcome in hiPSCs, which proved to be the most sensitive screening tool to identify toxicity. The results also demonstrate that long-term low-dose exposure of hiPSC-derived cardiomyocytes can be used as an in vitro model of delayed doxorubicin-induced cardiotoxicity and provide evidence that targeting GATA4 by small molecules may counteract doxorubicin-induced cardiotoxicity. Finally, the results reveal distinct PKC-dependent regulation of cardiac fibroblast transdifferentiation and proliferation and suggest that fibroblast-targeted PKC activation may be a promising strategy to inhibit cardiac fibrosis. Overall, the results highlight the importance of selecting an appropriate cell type and experimental model for compound testing and support further development of both GATA4-targeted compounds and PKC activators.
  • Tanner, Timo (Helsingin yliopisto, 2021)
    Developing new pharmaceuticals is costly and time-consuming. New methods are always in demand for various stages of product development. Investing in the early phases of development can save a significant amount of resources in the long term. Tablet is still the most commonly used pharmaceutical dosage form. Tablets are often produced by powder compression. Powder particles fragment and deform under pressure, allowing new bonds to form between them. Modern machines can produce over one million tablets per hour. The mechanical properties of the powders have a remarkable impact on compact formation. For example, excessive elasticity in a powder mixture can lead to weak or defected tablets being produced. Therefore, the mechanical properties need to be studied. Devices known as tableting simulators have been designed to aid in developing adequate tablet formulations. These machines are useful, but they can still be quite expensive and large. The results obtained by these machines are not always universally applicable, and further interpretation is often required. In this thesis, a novel gravitation-based high-velocity compaction (G-HVC) method was developed to study the compressibility and tabletability of powders in a cost-efficient and straightforward manner. The method is based on a freely falling steel bar, which compresses the powder sample inside a custom-made die. The movement of the bar and the deformation wave of the system base were monitored by high-accuracy displacement sensors. Displacement graphs could then be derived further. All data obtained by the method was ultimately only based on the displacement data. First, microcrystalline cellulose (MCC) and starch samples were compressed to demonstrate the functionality of the method. MCC was shown to be more compressible and less elastic than starch. Apparent differences in the relative volume decrease and the compression behaviour of these two materials could be seen. Next, various materials were studied more comprehensively. Two different setups with varying pressure were in use. Lactose grades and glucose showed effective fragmentation and reached true density with both setups. MCC grades were clearly pressure-dependent and showed slower gradual deformation, indicating plastic behaviour. Compression pressure was not high enough to effectively fragment calcium phosphate. Starch showed most elasticity of all the samples. In summary, all examined materials could be successfully categorized in terms of their mechanical properties. Finally, the practical relevance of the method was shown by creating a model between the compaction energy values determined by G-HVC method and the tensile strength of tablets produced with a tableting machine. Three different formulations consisting of MCC, calcium phosphate, theophylline and HPMC were granulated utilizing a fluid bed system. There was a good correlation between compaction energy and tensile strength. In summary, the G-HVC method was proven to be a reliable and cost-efficient tool in the examination of the mechanical properties of powders. The method was also capable of producing practically relevant results. The method fits well in modern pharmaceutical research where material-sparing, straightforward and reliable methods are in demand.
  • Hassan, Ghada (Helsingin yliopisto, 2021)
    Antibiotic resistance is a current threat to modern medicine. Not only is it challenging to treat but it also adds considerable costs to the healthcare systems. At the current rate of rising drug resistance, approximately 10 million people will die annually by 2050. The World Health Organization (WHO) has listed the six most threatening pathogens for which new antibiotics and approaches are urgently needed. One of the fastest evolving and most notorious Gram-positive bacteria in the list is Staphylococcus aureus. In addition to developing resistance to a vast number of antibiotics, this bacterium can attach to surfaces and form biofilms. This lifestyle allows bacteria to protect themselves from the immune system and impairs treatment. Thus, new innovative approaches and antibacterial agents are needed to fight bacterial resistance. As microbial adhesion is the first step of biofilm formation, the first aim of this study was to develop novel antibacterial surfaces to hamper the accumulation of antibiotic-resistant microbes. Cellulose is the most abundant natural polymer with various appealing characteristics. It is renewable, biocompatible, biodegradable, and possesses excellent mechanical properties. Cellulose nanofibers are prepared by mechanical disintegration of cellulose. Thin films of cellulose nanofiber can be prepared, and their surface can be modified to provide cellulose with new properties. Terpenoids such as abietic acid and dehydroabietic acid originating from conifer resin possess antibacterial and antifungal activities. We have previously shown that the amino acid-bearing derivatives of dehydroabietic acid possess antimicrobial properties. Herein, I report the design and the synthesis of new wide-spectrum contact-active non-leaching antibacterial cellulose nanofiber films by coupling the films with dehydroabietylamine, dehydroabietic acid and their derivatives. Different techniques and measurements were used to study the new biomaterials including contact angle (CA), streaming current measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), oxygen transmission rate (OTR), water vapor transmission rate (WVTR), scanning electron microscope (SEM), tensile strength, and Young’s modulus. Our unique design rendered four anionic surfaces highly active against the Gram-positive S. aureus, including the drug resistant methicillin-resistant Staphylococcus aureus (MRSA) and the Gram-negative Escherichia coli. The proposed modes of action and the fact that the compounds are based on a new chemical class account for a low potential to spread resistance. Our most active material 87 was tested against bacterial colonization in both biofilm and artificial dermis models. The bacterial colonization was efficiently prevented in both models. Material 87 proved to be biocompatible as it nurtured fibroblast growth at its surface without causing significant hemolysis. Our originally designed surfaces represent a new class of renewable biomaterials suitable for biomedical applications. The second aim of this thesis was to develop a new class of pyrimidine derivatives against S. aureus biofilms. From our novel set of pyrimidines, compounds 89, 99e, and 100e displayed potent activities. They inhibited biofilm formation and were active against pre-formed biofilms of S. aureus ATCC 25923 and Newman strains with IC50 values ranging between 11.6 to 62.0 μM. The compounds were also effective against planktonic cells with minimum inhibitory concentration (MIC) values lower than 60 μM. Only marginal cytotoxicity was revealed against human Hep2 cells at concentrations comparable to their pre-exposure IC50 values. Overall, this study resulted in the synthesis of four new antibacterial biomaterials and 26 new pyrimidine derivatives. This thesis offers novel approaches to target one of the most dangerous antibiotic-resistant bacteria S. aureus and efficiently limit its proliferation.
  • Kiiski, Iiro (Helsingin yliopisto, 2021)
    Drug metabolism is an enzyme-catalyzed process that has major implications for a drug’s safety and efficacy. Consequently, evaluating the metabolic properties of a new drug candidate is of paramount importance already in the preclinical phase of drug development. Although the available in vitro techniques for drug metabolism research have improved over recent years, the state-of-the-art methodology relies on enzyme assays conducted in static conditions, with limited spatiotemporal control of assay variables. Conducting metabolic assays under flow-through conditions could pave the way for more in-depth understanding of the mechanistic basis underlying drug-enzyme and drug-drug interactions. This, however, requires that drug-metabolizing enzymes be immobilized onto a solid support material without compromising protein folding or enzyme function. Although a wealth of different enzyme immobilization strategies are currently available, most of them are not amenable to immobilization of the microsomal, drug-metabolizing enzymes. The aim of this thesis was to establish immobilized enzyme microreactor (IMER) platforms for studying drug metabolism under flow conditions, thereby improving the in vitro-in vivo prediction of the metabolic fate of new drug candidates. The use of microfluidics and microfabrication technology facilitated the straightforward implementation of flow-through assays and furthermore allows their multiplexing (parallelism) and integration with other operational units on a single platform, minimizing both reagent consumption and dead volumes. In the first sub-project (publication I), a novel strategy for the immobilization of the membrane-bound enzymes cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) was designed and implemented on microreactors fabricated from off-stoichiometric thiol-enes (OSTE). The strategy was based on biotinylation of human liver microsomes via biotin-tagged fusogenic liposomes, and utilized the tunable surface chemistry of OSTEs to allow easy functionalization of the microreactor surface. The IMER platform was preliminarily characterized to ascertain enzyme stability and preservation of key enzyme kinetic parameters, with an emphasis on CYP-mediated (phase I) oxidoreductive reactions. In the second sub-project (publication II), the feasibility of the IMER platform for studying the kinetics of UGT-mediated drug conjugation (phase II) was assessed, with an emphasis on the mechanistic basis for the pronounced underestimation of in vivo clearance kinetics by the currently available static in vitro techniques. In particular, the effect of membrane disruption and fatty-acid inhibition on UGT-kinetics in vitro was studied in detail. The kinetics of zidovudine glucuronidation (the model reaction used in this study) under flow-through conditions was shown to be in good agreement with that obtained using microsomal incubations in static conditions without the need for added pore-forming agents such as alamethicin. In the third sub-project (publication III), the technology was further developed by incorporating a highly overlooked dimension in the assay design: the impact of oxygen partial pressure on the metabolic fate of drug candidates. This was achieved by exploiting the unique material-induced oxygen scavenging property of thiol-ene polymers. The developed chip design allowed the rapid and simple control of oxygen concentration in enzymatic assays, which is difficult to achieve with conventional static assay methods. Overall, the developed methodology was shown to retain enzyme activity and native enzyme kinetic parameters of both CYP and UGT enzymes, an unconditional prerequisite for drug metabolism assays. With the immobilization method utilizing membrane biotinylation via liposome fusion, common problems (such as diffusion-limited kinetics and enzyme inactivation) associated with conventional immobilization approaches were circumvented. Furthermore, the universal applicability of the immobilization method for all membrane-bound enzymes was preliminarily demonstrated with recombinant CYP enzymes in sub-project/publication I. The methodology developed here also enabled mechanistic studies focused on the alamethicin-induced membrane disruption (commonly used in UGT assays to overcome mass transfer limitations) and the proposed inhibitory effects of fatty acids, which may shed light on the foundations behind the poor in vivo correlation associated with UGT reactions in vitro. The material-induced oxygen scavenging facilitated by OSTE polymers, together with microfluidic actuation, was shown to be an easily controllable approach for adjusting the oxygen partial pressure on demand. The advantage of this approach is that, unlike in typical approaches, complex chip designs or pressurized compartments are not needed. Beyond the demonstrated feasibility of the IMER platform for studying the impact of NADPH-cytochrome P450 oxidoreductase (POR)-mediated metabolism, the theoretical framework established here for OSTE-enabled oxygen control in microfluidic assays is likely to find many applications, particularly in organ-on-a-chip research. In conclusion, the microreactor platform developed in this thesis offers an enabling toolbox for conducting in vitro drug metabolism assays under flow conditions that circumvents many of the key shortcomings of current state-of-the-art (static) in vitro enzyme assay methodology, as well as those of previously reported IMER platforms. At the time of publication, the methodology developed herein has already been utilized in several follow-up studies and has shown utility in diverse applications beyond this work.
  • Renko, Juho-Matti (Helsingin yliopisto, 2021)
    Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of nigrostriatal dopamine neurons and propagating Lewy body pathology. Dopamine depletion in the striatum gives rise to the cardinal motor symptoms of PD. Current PD medications are based on replenishing striatal dopamine and provide symptomatic relief to the motor deficits. However, troublesome adverse effects and diminished efficacy complicate their long-term use. There is a great unmet medical need for a therapy that could slow or halt the progression of the disease. Neurotrophic factors (NTFs) are secreted proteins that promote neuronal growth, differentiation and survival. They are able to prevent the progression of neurodegeneration and restore aberrant neuronal function in a variety of preclinical models of PD. Nonetheless, outcomes from clinical trials have been disappointing. The purpose of this work was to characterize the effects of cerebral dopamine neurotrophic factor (CDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF) and novel small molecule receptor tyrosine kinase RET agonists (BT13 and BT44) on nigrostriatal dopamine system and support their preclinical development as potential neurotrophic therapies of PD. To further clarify the functional effects of glial cell line-derived neurotrophic factor (GDNF), CDNF and MANF in the normal rat brain, microdialysis measurements were performed after a bolus injection of NTFs into the striatum. We saw augmented stimulus-evoked dopamine release and elevated dopamine turnover in the striatum of MANF-injected rats. GDNF injection increased in vivo tyrosine hydroxylase (TH) and catechol-O-methyltransferase activity and decreased monoamine oxidase A activity. These data are relevant when considering exogenously administered NTFs as a potential therapeutic approach for PD, since they have to be compatible with the existing dopaminergic medications of the patients. We also investigated the distribution properties of 125I-labeled and unlabeled CDNF after a nigral injection to intact rats. CDNF readily diffused into the brain areas surrounding the injection site and colocalized with TH-immunoreactive neurons in the substantia nigra. We did not detect active transportation of CDNF to distal brain areas. This characterization provides valuable insights into the selection of optimal delivery site and protocol for CDNF therapy. Our in vitro assays showed that RET agonists BT13 and BT44 were able to induce RET phosphorylation and activate downstream pro-survival signaling cascades Akt and ERK. They also supported the survival of cultured midbrain dopamine neurons from wild-type, but not from RET knockout, mice. The functional effects of BT13 and BT44 were evaluated in a unilateral 6-hydroxydopamine rat model of PD, where both compounds alleviated amphetamine-induced turning behavior. BT44 also showed potential to restore striatal TH-immunoreactive fibers. As blood-brain barrier penetrating compounds, BT13 and BT44 serve as promising leads that can be further developed into a disease-modifying therapy for PD.
  • Suutari, Teemu (Helsingin yliopisto, 2020)
    Cell-based assays have become an integral part of pharmaceutical research and drug development. They offer researchers not only a means to investigate basic cellular functions and different disease states, but also a way to study drug action in cells. Nowadays, during the long journey that new drug candidates take from an early discovery to a final product, all drug candidates undergo a preclinical test phase where their properties are evaluated using various cell-based assays. While perhaps the most common and important goal for these cell-based assays is to ensure that the investigated molecules are not harmful to cells, cell-based assays are also used to investigate the biological activity, mechanisms of action, and possible interactions, such as interaction with other drugs. With the traditionally used cell-based assays it is a common practice to alter the investigated pharmaceuticals or cells, or both, to produce a measurable signal, such as radioactivity, chemiluminescence, or fluorescence. However, there are well-known issues with this strategy, not the least that by subjecting the pharmaceuticals and/or cells to these modifications, an artificial system is created where the cells or pharmaceuticals under investigation are no longer in their original state. This can have a profound impact on the properties of the pharmaceuticals or behavior of the cells, thereby resulting in erroneous readouts. Because of this, different label-free cell-based assays are an attractive and logical alternative. Label-free technologies have been applied in the field of drug research and discovery since the 1980s. During the turn of the last millennium an increasing number of studies have implemented these methods to measure cellular activity, such as receptor activation. Nowadays, some of the commercial label-free devices are designed specifically with cell-based assays in mind. Of course, these technologies are not without their own challenges. The most prominent of these stems from the inherit property of these technologies: they are not sensitive to just a single predefined cellular event or response, but instead they measure cell activity as a whole. While well-controlled experimental design can mitigate the contribution of non-relevant cell responses to the measured label-free signals, advanced signal analysis methods have made, and can be expected to continue to make, label-free cell-based assays even more powerful tools for cell studies and help researchers to better understand the effect of different cellular activities on the label-free signals. Amongst the different label-free techniques, optical methods have been widely adopted for cell-based assays. While resonant waveguide grating (RWG) has been mainly utilized in the investigation of membrane receptors, namely G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), surface plasmon resonance (SPR) has been applied in the study of a variety of cellular processes, such as cell adhesion, detachment, spreading, contraction, cytoskeleton rearrangement, cell-to-cell contacts, monolayer formation and cell death. Surprisingly, studies on GPCRs with SPR are relatively low in number, even though they should be of high interest as around 35% of all current drugs mediate their effect via these receptors. Although SPR has already been shown to be a powerful tool for studying various cellular processes, its wider use in life sciences is hampered by the challenge in interpreting the contribution of the cell responses of interest and other non-relevant responses interfering with the measured signals. A variety of NPs are used as drug carriers, especially for biological drugs, in order to protect them from degradation and guide them to diseased cells or other desired location within the human body, while EVs are naturally produced nanosized structures that function as message carriers between cells. Because of the inherent property of EVs to penetrate cellular barriers, there is a growing interest in using them as “natural” and personalized drug carriers. This thesis investigates the use of SPR in the context of cellular uptake studies of nanoparticles (NPs) and extracellular vesicles (EVs), and GPCR-mediated signaling. Various approaches to analyze real-time label-free SPR signals are implemented in order to improve the correlation between the measured SPR responses and cell activities. The results show, for the first time, how SPR can be used to measure cellular uptake of NPs and EVs. However, because NP and EV uptake lead to ambiguous SPR signals, different signal analysis strategies are investigated and implemented to provide meaningful SPR responses. Three strategies proved to be effective: (1) varying the measurement temperature allowed to investigate possible cell entry mechanisms of different NPs in HeLa cells; (2) correcting the SPR angle response by removing the contribution of total internal reflection (TIR) angle shift allowed the quantification of the uptake efficacy of functionalized NPs into HT-29 colorectal adenocarcinoma cells; (3) comparing the responses measured with two different wavelengths corrected for the large response variation observed across different experiments for EV uptake in PC-3 prostate cancer cells. In addition, multiple label-free SPR parameters from the full SPR curves were analyzed after stimulating different GPCR subtypes, signaling primarily via Gs-, Gq- or Gi-coupled pathways. It was revealed that by combining two key label-free parameters, unique label-free signal profiles for each pathway could be generated, which makes it possible to recognize the pathway coupling of the receptor subtypes from the label-free responses alone, which has not been possible before. Collectively, these results demonstrate how SPR can be effectively utilized in cell-based assays to investigate NP and EV uptake and cell signaling. Cell-based SPR provides high information content, and with the help of the developed and used analysis methods, the otherwise hard-to-interpret label-free signals provide meaningful information on these cell responses.
  • Kortesoja, Maarit (Helsingin yliopisto, 2020)
    Chlamydia pneumoniae is an obligate intracellular human pathogen whose primary site of infection is the respiratory tract. In addition to respiratory tract infections, such as sinusitis and pharyngitis, C. pneumoniae has been related to several chronic inflammatory diseases of which atherosclerosis is the most widely studied. In order to contribute to the pathogenesis e.g. in atheromatous arteries, C. pneumoniae must disseminate from lungs to other tissues. This translocation occurs via peripheral blood mononuclear cells (PBMCs), mainly monocytes and macrophages. The presence of C. pneumoniae has been reported to induce inflammatory molecule production and alter redox balance in these cells, while also promoting their foam cell formation, migration and adherence. The ability of C. pneumoniae to persist inside the PBMCs, and thus become refractory to conventional antibiotics complicates the management of the associated underlying infection in chronic inflammatory diseases. Most C. pneumoniae susceptibility studies are currently based on the use of permissive cell lines, such as epithelial and endothelial cell lines in which the bacteria are actively replicating, which leaves the persistent infections unnoticed. Therefore, in addition to new active compounds against C. pneumoniae, new methods to study their effects against the persistent chlamydial infection are urgently needed. In this study, dibenzocyclooctadiene lignans, new antichlamydial compounds originating from berries of medicinal plant, Schisandra chinensis, were profiled regarding different aspects of C. pneumoniae-PBMC interactions. Human THP-1, a monocyte and macrophage cell line, and a murine macrophage cell line RAW264.7 were used in the studies to examine the effects of the schisandrin lignans on C. pneumoniae infection and their influence on host cell responses. Oxidative stress is the basic pathological mechanism underlying a spectrum of chronic inflammatory diseases, and it largely contributes to the consequences of C. pneumoniae infection as well. In this work, C. pneumoniae was found to induce proinflammatory cytokine, interleukin (IL)-12 secretion in the human monocytic cell line. It also increased the intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels in macrophages, and it had an impact on the concentration of glutathione (GSH), the major small-molecule antioxidant, in macrophages. Gene expression analysis of murine macrophages revealed that C. pneumoniae suppressed the peroxisome proliferator-activated receptor γ (PPARγ) transcription in the cells, which influences lipid metabolism and inflammatory responses in these cells. Schisandrin lignans had an impact on C. pneumoniae-induced alterations in these cell models. Schisandrin B and schisandrin C reduced the elevated interleukin (IL) -12 cytokine levels, as well as the LPS induced IL-6 and IL-12 levels. Schisandrin lignans also affected cellular oxidative balance by elevating the basal ROS levels while simultaneously reducing the ROS or NO levels induced by infection or LPS. The redox balance alteration was also shown within the GSH levels, which were reduced by the lignans in THP-1 monocytes and macrophages but elevated in RAW264.7 cells. Schisandrin B additionally upregulated the transcription of genes involved in GSH synthesis, GCL and GGT-1. Schisandrin B and schisandrin C also reduced the C. pneumoniae-induced macrophage foam cell formation and altered the related expression of PPARγ and ABCA1 genes. In this work, a new platform for studying the C. pneumoniae transfer between lung epithelial cells and phagocytes, is also introduced. The platform can be used in lead compound profiling studies against C. pneumoniae infection. Mitogen-activated protein kinase (MAPK) inhibitors were found to inhibit the transfer of the infection, serving as reference compounds in future profiling studies. This work provides new information about the C. pneumoniae infection in monocyte-macrophage cell models, while also offering valuable insight on the antichlamydial lead compounds, dibenzocyclooctadiene lignans. These lignans were shown to suppress the C. pneumoniae induced pathological changes in the host cells. With newly identified antichlamydial activities and novel study methods, we can explore more impactful approaches to overcoming C. pneumoniae-induced chronic inflammatory diseases.
  • Kari, Otto (Helsingin yliopisto, 2020)
    Nanoparticles administered into the body are rapidly covered by biomolecules and assume a biological identity that mediates their biological interactions. Methodological constraints have limited our ability to study the formation of this “protein corona”. To increase our understanding of its formation and evolution in different biological environments, and hence improve the safety and efficacy of nanomedicines, there is a need for non-invasive and label-free methods that better mimic the conditions inside the human body. This thesis describes the development of a novel label-free workflow to determine the structure and composition of the hard and soft corona on liposomes. Multi-parametric surface plasmon resonance is adapted to probe corona structure without interference, including the soft corona of loosely bound proteins. Microfluidics is used to elute the corona subsections for proteomics analysis of their compositions by nanoliquid chromatography tandem mass spectrometry. The in vivo relevance of the method is improved by the use of undiluted biological fluids under dynamic flow conditions. The workflow was applied to the preclinical development of pH-responsive and light- activated liposomes intended for tumour targeting and ocular drug delivery. A novel hyaluronic acid (HA)-coated liposome was also synthesized. The results indicate that thin and sparse monolayer hard coronas form on the liposomes in plasma and vitreous. It is covered by a soft corona of monolayer thickness in plasma, but in vitreous, the soft corona is less defined. Formulation-dependent differences in corona structure and relative protein composition were observed in plasma, and the liposomes clustered based on their surface charge. In vitreous, corona composition was independent of formulation properties. In both environments, there is a high overlap in hard and soft corona compositions, and the most abundant proteins are predominantly hydrophilic and negatively charged irrespective of liposome properties. A common set of surfactant and immune system associated proteins adsorbs in both plasma and vitreous, possibly in response to the high surface free energy of lipid bilayers, labelling them “non-self” lipid particles. However, liposomal lipids induced the enrichment of stealth-mediating proteins in the plasma hard coronas regardless of pegylation. These also enriched in the soft coronas of most other formulations, suggesting that the soft corona is part of the biological identity and modulated by liposome surface properties. Protein-specific, rather than formulation- specific factors, are drivers of protein adsorption in the vitreous, possibly due to the molecular crowding activity of structural HA. Although the HA-coated liposome bound more proteins in vitreous, which may interact with its structural meshwork, corona formation did not significantly influence the vitreal mobility of HA or PEG-coated anionic liposomes. The HA-coating yielded improved plasma stability of the light-activated liposomes with otherwise comparable properties, making it a promising coating for both intravenous and ocular drug delivery applications. The workflow addresses significant methodological gaps in the research field by providing information on truly complementary hard and soft corona compositions together with their in situ structural properties. This is achieved with undiluted biofluids and under dynamic conditions without the use of interfering labels. The first description of vitreal corona formation on drug delivery systems contributes to our understanding of ocular pharmacokinetics. The method was also applied to determine the kinetics of opsonin binding directly on liposomes for the first time. The convenient and easily reproducible workflow is well-suited for the preclinical development of liposomes, and it can be combined with other omics methodologies and adapted to accelerate the preclinical development of different types of nanomedicines.
  • Rosenholm, Marko (Helsingin yliopisto, 2020)
    Anesthetics are commonly used to induce unconsciousness and insensateness during surgery. However, the impacts of anesthetics on brain function go well beyond their acute pharmacological effects. Animal research suggests that the developing brain is particularly vulnerable to anesthesia, and even a single exposure may induce persistent neurobiological and behavioral consequences. Nevertheless, anesthetics have demonstrated remarkable therapeutic potential against some prevalent and debilitating brain disorders, especially major depression. Indeed, a single subanesthetic dose of ketamine has been reproducibly shown to alleviate depression and suicidal thinking within hours of administration, and the effects can last for days. Induction of brain-derived neurotrophic factor (BDNF) receptor TrkB signaling and synaptic plasticity have been intimately connected with ketamine’s antidepressant effects, but the precise mechanistic basis remains obscure. Notably, rapid antidepressant effects have also been reported with other anesthetics, including nitrous oxide (N2O) and isoflurane, and after somatic treatments such as electroconvulsive therapy (ECT) and sleep deprivation. In the first part of this thesis, we investigated the long-term behavioral effects of early postnatal exposure to repeated brief isoflurane anesthesia. We exposed mouse pups to anesthesia on three consecutive days at two distinct developmental stages, at postnatal days 7–9 or 15–17, and later tested the behavioral phenotype of the adult animals. Isoflurane anesthesia caused modest behavioral effects on locomotor activity and spatial learning and memory of the adult mice, depending on the age of the animals during the anesthesia exposures. In the second part, we investigated the effects of various anesthetics on depressive-like behavior and molecular signatures connected to the antidepressant effects of ketamine in rodents. We subjected rats to the chronic mild stress model of depression and subsequently exposed them to repeated brief isoflurane anesthesia for a total five times every three days. This administration regimen, however, was insufficient to normalize anhedonic behavior in the stressed rats. Cortical and hippocampal BDNF levels in these rats also remained unaltered. We then investigated the dose-dependent and temporal effects of different anesthetics on TrkB signaling, activity-dependent immediate-early genes (IEGs), and electroencephalographic (EEG) activity in mice. Here, we discovered that N2O upregulated several IEGs (markers of cortical excitation) during acute pharmacological effects, and that these effects were followed by a rebound emergence of EEG slow-wave activity (SWA) and TrkB signaling after treatment cessation. Similar concurrent upregulation of SWA and TrkB signaling was evident after a flurothyl-induced seizure (reminiscent of ECT) and during the effects of a sedative drug, medetomidine. Medetomidine, however, lacked antidepressant-like effects in the learned helplessness model of depression. This suggested that instead of only an increase in SWA and TrkB signaling, a preceding excitatory effect is also crucial for rapid antidepressant effects. This may also explain our observed lack of behavioral effects of deep isoflurane anesthesia. Moreover, even though ketamine’s antidepressant effects are associated with subanesthetic doses, we found that ketamine increases SWA and TrkB signaling, with the most pronounced effects observed at high anesthetic-sedative doses. These effects appear independent of hydroxynorketamine, an active metabolite of ketamine that has demonstrated antidepressant-like effects in rodents. In conclusion, we found subanesthetic ketamine, N2O, and flurothyl to induce SWA after their acute pharmacological effects subsided. Interestingly, this phenomenon resembles the well-known postictal (i.e., after seizure) slowing of EEG activity, which has been connected to the antidepressant effects of ECT. Furthermore, the emergence of SWA coincided with the upregulation of TrkB signaling. Based on our results, we propose that rapid-acting antidepressants induce two distinct phases in the brain, with an initial excitatory phase followed by a sedative-like brain state that is characterized by SWA and TrkB signaling. Further studies are needed to elucidate whether a similar phenomenon is shared by other treatments that have demonstrated rapid antidepressant effects (e.g., isoflurane, psilocybin). The current proposal provides a novel framework for future research that encourages expanding the research focus from the acute pharmacology of the treatments to homeostatic alterations that potentially emerge as an intrinsic response of the brain to a drug challenge.
  • Palomäki, Emmi (Helsingin yliopisto, 2020)
    Drugs must dissolve upon administration to have a therapeutic effect. Nowadays, most new drug candidates are poorly water-soluble, which makes this solubility issue a significant global challenge. Solubilization can be enhanced using formulation-based solutions, particle size reduction, salt formation, prodrugs or amorphization of the drug. This thesis concerns the last approach, amorphization. Unlike highly ordered crystalline materials, amorphous materials lack long range order. This leads to amorphous materials having greater molecular mobility and free energy, and consequently solubility, than their crystalline counterparts. However, the solubility benefits of the amorphous form come with a price, since the thermodynamic instability of amorphous materials means they tend to crystallize. Pharmaceutical products need to be sufficiently physically and chemically stable throughout their entire shelf life to ensure their efficacy and safety. In the case of amorphous drugs, there are still many aspects about crystallization that are not fully understood. The aim of the thesis was to investigate factors influencing the crystallization process in single and multiphase amorphous systems, as well as complexities in monitoring the progression of crystallization. The crystallization of several one- and two-phase amorphous systems were investigated, with the influence of both excipients and atmospheric gas on the crystallization process being investigated. Raman spectroscopy and X-ray diffractometry were used to monitor crystallization in the study, and their sensitivities and suitability to measure crystallization in the samples of interest were considered. Differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and optical microscopy were used to provide complementary information on crystallization processes. In the present thesis it was found that excipients and atmospheric gases that interact with amorphous material, even in the absence of mixing and specific interactions, can delay the onset of crystallization. Additionally, it was found that Raman spectroscopy is not necessarily suitable for crystallinity determination, when the opacity of the sample changes, as can happen at temperatures above Tg. Overall, this thesis demonstrates that several factors, beyond those usually considered in traditional single-phase solid dispersions, can influence crystallization, and that these, together with the effect of measurement technique artefacts, should be carefully considered when developing amorphous formulations.
  • Sinnemäki, Juha (Helsingin yliopisto, 2020)
    Polypharmacy, i.e. concomitant use of several drugs is common among older adults. This increases the risk of using drugs that are potentially inappropriate and harmful for geriatric patients. Automated dose dispensing (ADD) is a procedure that has been implemented in some European countries, particularly in the Nordic countries and the Netherlands to manage these risks in primary care. In the ADD service, regularly used medicines are machine-packed into unit-dose pouches according to administration times. The service is expected to enhance appropriate drug use and to prevent medication-related harm among older adults as well as to decrease medication costs, and save nurses’ working time in primary care. This doctoral study aimed to investigate the existing evidence on the outcomes of the ADD service, assess the service’s initiation process and evaluate its impact on drug use and quality. A systematic literature review was conducted to summarize the existing evidence on the outcomes of the service in primary care. The initiation process of the ADD service was investigated by surveying community pharmacies offering the service. The service’s impact on drug use and quality were investigated using a retrospective cohort study with matched controls applying nationwide register data. The literature was systematically reviewed until the end of 2019. 20 studies were included, and only two of them were controlled intervention studies exploring the outcomes of ADD in primary care. Consequently, the evidence for ADD’s impact on appropriateness and safety of medication use is limited, and lacking on economic outcomes. When the ADD service was initiated, the medication list was incomplete for more than half (63%) of the patients (n=147). Community pharmacists collected information on patient’s medication from multiple sources to reconcile the list. Some type of medication review was conducted for most (96%) of the patients when the ADD service was initiated for them. Most commonly (69% of the patients) it was a prescription review, which is the least comprehensive type of medication reviews. Medication-related therapeutic changes were implemented for almost half (43%) of the patients, and almost all (93%) had technical changes due to the ADD process requirements in their medications while initiating the service. The retrospective register-based controlled study revealed that ADD users (n=2073) had more starts and discontinuations in their medications compared to their matched controls (n=2073). The results also suggest that drug use was decreased after the ADD service was initiated. When the quality of drug use was assessed by explicit criteria for potentially inappropriate medications for older adults (PIMs by Beers criteria 2012), an improvement was found. However, more complex problems in the drug regimens could not be solved. When the quality of drug use was assessed with more complex criteria, such as concomitant use of three or more psychotropic drugs, the quality of drug regimens was not improved. The results of this study imply that medication reconciliation and review need to be integrated into the ADD service procedure as an essential part of it. Both information technology systems and processes in healthcare organisations need to be further developed to ensure that medication records and lists are up-to-date. More comprehensive medication review than prescription review needs to be implemented as a part of the ADD service procedure to ensure rational pharmacotherapy for the ADD users. When municipalities and healthcare providers are purchasing ADD services, medication reconciliation and review need to be included as part of the contract.
  • Gatta, Viviana (Helsingin yliopisto, 2020)
    To overcame the spread of bacterial resistance to traditional antbiotics, great interest has arisen towards antivirulence agents, compounds targeting virulence factors. In fact, as there is no link between growth and virulence, antivirulence agents are considered less prone to promote resistance development. In this context, quorum sensing (QS), a communication strategy among bacteria which regulates several bacterial functions including virulence, has been widely investigated for the development of QS inhibitors with the aim of limiting bacterial virulence. This study describes the development of a new assay for the discovery of inhibitors targeting LsrK, a key kinase for autoinducer 2 (AI-2) mediated QS establishment in enteric bacteria. LsrK in fact phosphorylates the AI-2 which, only in the phosphorylated form, can bind to the LsrR repressor and enhance the response to QS signals via activation of the lsr operon. The new assay was used for the screening of three different compound libraries. The best hits from the three campaigns were harpagoside and rosolic acid, presented in Study I, also active in cell-based AI-2 mediated QS assay. Additionally, the active compounds found in Study II and III provided interesting information about the catalytic site of LsrK. To facilitate the confirmation of hits selected by target-based assay and to offer a new tool for the rapid identification of QS inhibitors, Study IV describes the design, optimization and application of a new bioreporter strain, emitting luminescence as response to AI-2 mediated QS activation. The assay was used to test a set of 91 compounds selected to target the ATP binding site of LsrK. The same set of compounds was also tested in the target-based LsrK inhibition assay. The combined results led to the identification of 6 compounds, active in both assays, which thus may decrease response to QS by inhibiting LsrK. Additionally, 18 compounds were active only in the cell-based assay implying that they target other components of the pathway. These findings broaden our knowledge on LsrK and may be used as scaffolds to design compounds with improved properties. Furthermore, the AI-2 mediated QS interference assay represents an additional tool for the identification of QS inhibitors alone or in combination with target-based assays.
  • Parkkila, Petteri (Helsingin yliopisto, 2020)
    Lipids self-assemble into lipid bilayers, which divide bodily tissues into cells and into functionally specified compartments. Imbalances in the lipid composition and metabolism take part in severe neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. These conditions are currently only symptomatically treated, and the functional insight into the effects of the drugs in different stages of the conditions is lacking. In the field of pharmacy, the current drug design protocols rely on the separate evaluation of the binding affinity between target protein and drug and the extent of lipid bilayer permeation, which dictates how likely the drug is to reach its target. For example, the direct treatment of the central nervous system requires the drug to cross the blood-brain barrier. Most of the drug target proteins are, however, permanently attached to the lipid membranes. This thesis hypothesizes that lipids and proteins can act together in relation to drug action, which chemical variations in the membrane constituents can profoundly affect. Oxidative stress induces peroxidation of unsaturated fatty acids, modulating the membrane properties, such as the permeability to water. It is of importance to understand how lipid peroxidation influences the extent of segregation of the domains in the lipid membrane. Although the functionality of the domains \emph{in vivo} is elusive, they may be associated with a myriad of cellular functions, such as the attachment of the actin cytoskeleton. By measuring lateral diffusion of lipids in Langmuir monolayers, we showed that the presence of oxidized lipids could irreversibly modulate the miscibility of the segregated microscale domains. This may affect the action of proteins, drugs and other molecules that interact with such structures. Since biological membranes are incredibly complex, consisting of thousands of molecules, they are challenging to study. Therefore, model lipid membranes are used in membrane research. In this thesis, these model systems were characterized using label-free surface-sensitive analytical techniques. Using the membrane models, we showed that the membrane-bound catechol-\emph{O}-methyltransferase (COMT) is able to function at the membrane-water interface, suggesting that the membrane partitioning and orientation of its substrates and inhibitors influences the drug efficacy. Inhibition of membrane-bound COMT is desirable in Parkinson's disease since it elevates dopamine levels in the brain. Also, the inhibition prevents the methylation of levodopa, a dopamine precursor that is currently the primary therapeutic agent. Therefore, we studied the partitioning of dopamine and different catechol compounds to model lipid membranes. Partitioning to the membranes, where the existence of nanoscale domains is proposed, was limited. The partitioning also seemed to be modulated by the charge, lipophilicity and hydrogen-bonding capacity of the compounds and surface charge of the membrane. These factors also affect the orientation of the compounds in the lipid membrane, which can define the probability of the to-be-catalyzed moieties to reach the catalytic site of the protein. To conclude, the results of this thesis demonstrate that the lipid environment can modulate drug action, which may have consequences for the design of novel therapeutics for neuropathological conditions.
  • Itkonen, Jaakko (Helsingin yliopisto, 2020)
    Since their introduction in the late 20th century, therapeutic proteins have become an irreplaceable class of pharmaceuticals and are today used to treat a wide variety of diseases ranging from arthritis, diabetes, and various cancers to more recently, for example, asthma and migraine. In ophthalmology, the treatment of certain neurodegenerative diseases of the retina, such as age-related macular degeneration and diabetic retinopathy, has been revolutionized by therapeutic proteins that combat the pathological growth of abnormal blood vessels in the retina. As retinal diseases are some of the leading causes of vision loss and blindness globally, and expected to grow in prevalence with aging populations, the importance and need for such ophthalmologic therapeutic proteins is expected to increase. During the early development of therapeutic protein candidates, the production of functional protein in adequate amounts can often be a significant roadblock. In this thesis, the expression of soluble recombinant human ciliary neurotrophic factor (rhCNTF) – a neuroprotective protein with therapeutic potential against retinal neurodegeneration – in Escherichia coli was enhanced. Codon optimization of the hCNTF gene was combined with screening of different culture media, culture conditions, and fusion partners to pinpoint ideal expression conditions for rhCNTF. Following expression in the determined optimal conditions, the protein was purified with immobilized metal-ion affinity chromatography and gel filtration, and the in vitro activity of purified rhCNTF was demonstrated in a binding assay with its cognate receptor, CNTFRα. Overall, an 8–9 fold increase in soluble rhCNTF fraction and a 10–20 fold increase in yield was achieved, whereas earlier efforts to produce CNTF have commonly required purification from insoluble inclusion bodies and/or yielded low protein amounts. Furthermore, such a combinatorial approach is successful as a screening strategy for soluble expression and could be applied to other proteins of pharmaceutical interest. When taken out of their natural biological milieus, most proteins are only marginally stable and susceptible to environmental perturbations. As such, the formulation of a therapeutic protein aims to protect the protein and retain its stability and biological activity, and ultimately to guarantee the therapeutic efficacy and safety throughout the lifetime of the pharmaceutical. Here, further characterization, formulation, and stability studies were carried out with purified rhCNTF. The proper folding of purified rhCNTF was observed with circular dichroism spectroscopy and the biological activity of the protein was verified in a cell proliferation study with a CNTFα expressing cell-line. After screening, two buffers were chosen as storage buffers for rhCNTF. Whereas minute changes in rhCNTF’s oligomeric status were observed in only of these buffers, no changes in rhCNTF’s thermal stability were observed in either buffer during the study period. As such, these results provide a basis for further formulation development for rhCNTF. Although intravitreally injected therapeutic proteins have become the cornerstone in the management of retinal neovascularization, how and to what extent these and other proteins penetrate into the retina remains poorly understood. Here, permeation into the neural retina was observed with fluorescently labeled rhCNTF in ex vivo retinal explant models. Our results indicate that permeation to the CNTF-responsive target cells in the retina is not a limitation to exogenous CNTF’s direct neuroprotective actions. Moreover, our results provide further impetus to utilize ex vivo methods to systematically elucidate the retinal permeation and ocular pharmacokinetics of therapeutic proteins by and large. Therapeutic proteins have not only held the helm of best-selling pharmaceuticals for some time now, but currently also represent more than 40% of new pharmaceuticals in the development pipeline. Regardless, protein drug development has been plagued with ever increasing development costs yet with fewer new drugs entering the market, and there is an urgent call to disrupt this unsustainable cycle. While reasons for failure are diverse, for therapeutic proteins the most reported are poor therapeutic efficacy and immune responses, issues which are often encountered relatively late during the drug development workflow. Therefore, it would be of utmost utility to develop methods for detecting such susceptibility before significant effort and funds are spent in the development of less than ideal candidates. In this thesis, by integrating cell-free protein synthesis in small volumes together with split-intein mediated capture and light-triggered release, a streamlined platform for rapid protein production and screening was developed. Our results provide a proof-of-principle, with successful capture and release of protein of interest, as well as protein bioconjugation achieved using hCNTF as a model protein. The developed platform can be used for the rapid screening of therapeutic protein candidate producibility, and acts as a first module to be coupled to in-line assays for monitoring e.g. the candidate’s immunogenic potential, enabling such issues to be addressed/resolved already during early development stages.  
  • Toivo, Terhi (Helsingin yliopisto, 2020)
    Over the last decade, a great deal of research has described the medication safety risks in hospitals and institutional care both in Finland as well as globally. Less attention has been paid to the safety of medicine use in outpatient care, even though majority of the use occurs at home. The aim of this study was to enhance prospective medication risk management in outpatient care, by enhancing coordination of care with community pharmacists’ participation and use of risk management screening tools available. Specific objectives of studies I–III were: I) to demonstrate how community pharmacies can utilize their prospective surveillance system for screening clinically significant drug-drug interactions (DDIs) in outpatients and assess the rate of DDIs in a large national prescription sample. II) To integrate risk assessment tools, procedures and databases available in Finland to form a coordinated medication management model (CoMM) for older home clients involving home care nurses and practical nurses (PNs), physicians and community pharmacists. III) To assess the impact of the CoMM on medication risks identified in drug regimens of older home care clients over a one-year period. Medication risks assessed related to potentially inappropriate medications (PIMs), excessive use of psychotropics, anticholinergic and serotonergic load, as well as clinically significant DDIs. In study I, all DDI alerts issued by the online surveillance system were collected during a one-month period in 16 out of 17 University Pharmacy outlets in Finland, covering approximately 10% of the national outpatient prescription volume. The surveillance system was based on the FASS database, which categorizes DDIs into four classes (A–D) according to their clinical significance. Potential DDIs were analyzed for 276,891 dispensed prescriptions and they were associated with 11.2% of the prescriptions. Clinically significant DDIs categorized as FASS classes D (most severe, should be avoided) and C (clinically significant but controllable) were associated with 0.5% and 7.2% of the prescriptions, respectively. Studies II–III were conducted in primary care in the city of Lohja, Southern Finland. Health care units involved were the home care, public primary healthcare center and a private community pharmacy. System-based risk management theory and the action research method were applied to construct the collaborative procedure utilizing each profession’s existing resources in medication risk management of older (>65 years, n=191) home care clients. Study II produced a 5-stage medication management model (CoMM) suitable for screening medications of a high number of home care clients and identifying clients with potential clinically significant drug-related problems (DRPs). The core of the model was the triage meetings that proved to be a feasible method for customizing comprehensiveness of collaborative medication reviews, according to their clinical needs while minimizing physicians’ time demands. In study III, an RCT study design was used to assess the impact of the CoMM on medication risks identified in drug regimens of older home care clients over a one-year period. Participants’ (n=129) mean age was 82.8 years, 69.8% were female and mean number of prescription medicines in use was 13.1. The intervention did not show an impact on the medication risks between the original intervention group and the control group in the intention to treat analysis, but the per protocol analysis indicated a tendency for effectiveness, particularly in optimizing central nervous system medication use (benzodiazepines). Half (50.0%) of the participants with a potential need for medication changes, agreed on in the triage meeting, had none of the changes actually implemented. Study I demonstrated that community pharmacists can actively contribute to DDI risk management and systematically use their surveillance systems for identifying patients with clinically significant DDIs. In study II, the developed care coordination model (CoMM) was feasible for screening and reviewing medications of a high number of older home care clients in order to identify clients with severe DRPs and provide interventions to solve them, utilizing existing primary care resources. In study III, the CoMM intervention indicated a tendency for effectiveness when implemented as planned, particularly in optimizing CNS medication use during a 12-month follow-up. Our study revealed that organizations and health care units involved in home care clients’ medication therapy are currently working independently in silos, where no specific team membertakes holistic responsibility for medications. This study demonstrated the challenges to overcome when trying to change clinical practice and improve coordination between units involved in medication management of home care clients. Even though the outcomes of the intervention were not optimal, the value of the study is in discussing the real-world experiences and challenges of implementing new practices in home care. This study indicated that practitioners in Finnish health care are not well acquainted with systems thinking, a fact which needs to be addressed in the future. Further studies are needed on care culture and other contributing factors to high prevalence of PIM use and other risks for clinically significant DRPs identified in this study. Particularly, further investigation is needed on system-based factors contributing to situations where identified preventable clinically significant medication risks are left unsolved, as well as the relationship between inappropriate medication use and medication errors. A need for the organizational and national development of medication safety in primary care was identified in this thesis, which is line with the national and international publications, policy documents and recommendations. Furthermore, community pharmacists’ contribution to medication safety, particularly in older adults, should be better utilized in the future, as this thesis shows promising demonstrations. KEYWORDS: Medication risk management, medication-related risk, drug-drug interaction, primary care, home care, older adult, community pharmacy  
  • Sathyanarayanan, Gowtham (Helsingin yliopisto, 2020)
    Drug metabolism is a detoxification process by which the body converts pharmaceuticals into more hydrophilic metabolites. Understanding of the drug metabolism process and metabolic profiling plays a vital role in drug development processes by ensuring the safety and efficacy of treatments. Cytochromes P450 (CYP) are a superfamily of enzymes that are primarily responsible for metabolizing the majority of clinically relevant drugs. In preclinical drug development research, there is a constant need for the identification of metabolites and their CYP isoenzyme-specific elimination route, as well as possible drug-drug interactions thereof using high speed in vitro techniques. Miniaturization of the drug metabolism assays and related processes could further improve the throughput via parallelism and integration of several analytical steps on a single platform, as well as reducing the consumption of expensive reagents substantially. Micro total analysis systems (µTAS) usually refer to microfabricated devices that integrate several analytical unit operations, such as sample preparation, extraction, separation, and analysis on a single platform. These µTAS platforms can be either continuous-flow microchannel based systems or discrete droplet systems. Digital microfluidics (DMF) is one such technology, where sample droplets are manipulated individually on an array of electrodes. In DMF, the droplets of hundreds of nanolitres to a few microliters of volume can be dispensed, split, mixed, and merged independently via programmed and automated voltage application. In this thesis, several DMF-based bioanalytical concepts were developed and their feasibility for implementing droplet-scale drug metabolism assays was evaluated. In the first sub-project, droplet-scale immobilized enzyme reactors were developed by immobilizing CYP enzymes on porous polymer monoliths affixed onto a DMF platform. Assay incubation at physiological temperature was facilitated by localized heating of the DMF platform using integrated inkjet-printed microheaters. For the on-chip detection of drug metabolites, a protocol facilitating interfacing of the DMF device with a commercial wellplate reader was developed. In the second sub-project, the developed DMF platform, featuring the CYP reactors, were interfaced with ambient mass spectrometry (MS) via desorption atmospheric pressure photoionization (DAPPI). For in situ identification of the drug metabolites by DAPPI-MS, the chip design was optimized to be able to control the critical surface sensitive processes, such as sample precipitation and subsequent desorption/ionization directly from DMF surfaces. In addition, the feasibility of the same platform for a droplet-based liquid-liquid extraction of pharmaceuticals was demonstrated. All pharmaceuticals and metabolites analyzed could be detected with lower limits of detection in the range of a few picomoles. In the third sub-project, DMF droplet manipulation was interfaced with channel microfluidics to facilitate more versatile sample preparation such as separation of target analytes after the droplet-based enzyme reactions and prior to detection. To support the scaling up of the developed technology toward mass manufacturing, the entire device was assembled using low-cost inkjet printing and non-cleanroom polymer processing techniques. To achieve this interfacing, off-stoichiometric thiol-ene (OSTE) polymers were introduced as a new alternative dielectric material for the coating of inkjet-printed DMF electrode arrays, as well as for the integration of the microchannels with a DMF platform. In the fourth sub-project, magnetic bead based enzyme immobilization protocol was developed to facilitate screening the individual variation of CYP activities in donor-derived human liver microsomes (HLM) in droplet-scale. A CYP1A isoenzyme-specific model reaction was chosen to assess the inter-individual variation in the activities of this metabolic route in the liver microsomes collected from five individuals. The demonstrated protocol was shown to be technically feasible for biopsy-scale samples. In all, the new droplet-scale concepts developed in this thesis are first-in-their-kind examples of droplet-scale drug metabolism assays on DMF platform. The methods developed are generally qualitative or semi-quantitative and thus, in their present form, best feasible for the preliminary determination of metabolic clearance via CYP or identification of the produced metabolites of new drug candidates in vitro. Further development of the technology, particularly the enzyme immobilization process and the quantification of the produced metabolites, is needed to improve the wider applicability of the assays. It is noteworthy however that all of the fabrication processes and interfacing approaches taken in this thesis were carried out in regular, non-cleanroom laboratory conditions, which is foreseen to significantly improve the adaptability of the technology in any bioanalytical laboratories.
  • Lillsunde, Katja-Emilia (Helsingin yliopisto, 2020)
    Viruses are accountable for numerous diseases that form an immense threat to public health worldwide. The capability of viruses to continuously adapt to a changing environment makes the discovery of new treatments for viral diseases crucial. Natural products play an important role in the discovery of new drug candidates, essentially as a source of lead compounds that can be chemically optimized to achieve improved drug properties, such as safety and efficacy. In natural product drug discovery, the marine environment offers outstanding potential to discover new compounds with interesting bioactive properties. Marine species and their unique metabolites are still only partly discovered, and at the same time, vulnerable marine environments are threatened by human activities through pollution, overexploitation and climate change. The protection of marine biodiversity and sustainable use of marine resources is therefore a vital part of the study of marine organisms and their metabolites. This dissertation focuses on studying the antiviral properties of marine-derived compounds and their synthetic derivatives. The first part of this study covers screening of crude extracts from the Indian Ocean soft coral Sinularia kavarattiensis in a chikungunya virus replicon model followed by bioactivity-guided isolation and further study of the purified compounds. This study led to the isolation of six known norcembranoid compounds and the isolation and characterization of one novel compound, kavaranolide. Two of the isolated compounds were moderately active in the chikungunya replicon model, but also showed cytotoxic properties. The second part of the research focuses on studying the antiviral potential of synthetic compounds inspired by the marine sponge-derived alkaloids clathrodin and oroidin. In the screening of a compound library of 157 clathrodin and oroidin analogues in chikungunya virus and hepatitis C virus replicon models, four compounds were discovered to selectively inhibit the hepatitis C replicon with IC50-values ranging from 1.6 to 4.6 µM. Interaction with the cellular chaperone Hsp90 was proposed as the mechanism of action underlying the activity, and this hypothesis was supported by the results from molecular modelling and microscale thermophoresis interaction studies. Based on the study of clathrodin and oroidin analogues, 12 new compounds with a 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole structure were synthesized, in order to obtain improved binding to Hsp90 and improved antiviral properties. Three of the synthesized compounds showed improved binding to Hsp90 and specific inhibition in hepatitis C genotype 1b and 2a replicon models and moreover, inhibited the replication of full-length hepatitis C genotype 2a virus in a reporter virus RNA assay (IC50-values 0.03–0.6 µM). As Hsp90 is a host protein utilized by the viral replication machinery, antiviral activity achieved through inhibition of Hsp90 could be an attractive strategy to combat resistant viral strains.

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