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  • Suotunen, Pauliina (2020)
    The OATP1B3 belongs to the organic anion transporting polypeptides (OATPs) encoded by the SLCO (solute carrier organic anion) genes which belongs to the SLC (solute carriers) gene superfamily. It is an influx transporter which is primarily expressed on the basolateral membrane of the hepatocytes. It transports many endogenous substrates as well as clinically important drugs such as thyroid hormones and statins into hepatocytes and thus participates in the first step of hepatic metabolism. Single nucleotide polymorphisms (SNPs) of the SLCO1B3 gene can affect the pharmacokinetics and pharmacodynamics of its substrates. The aim of this study was to set up and optimize an in vitro method to study the function and expression of the OATP1B3 transporter and its genetic variants. SNPs 334T> G (Ser112Ala), 699G> A (Met233Ile) and 767G> C (Gly256Ala) and stop codon TAA were introduced into the SLCO1B3 gene by site-directed mutagenesis. Recombinant baculovirus vectors containing the genetic information of OATP1B3 and its variants were used to transiently transfect the HEK293 cells. After optimizing the substrate incubation time and concentration, as well as the viral load and selecting the fluorescent substrate (8-FcA), the uptake assay was used to determine the transport activity of the OATP1B3 variants in HEK293 cells. The transport activity of the artificial WTP variant was also investigated in this study. The transport activities of the Ser112Ala, Met233Ile and Gly256Ala variants did not change significantly from the wild type although the transport activity of the Met233Ile variant appears to be slightly impaired. In turn the WTP variant was unable to transport 8-FcA. Based on this study the function of OATP1B3 variants can be studied using recombinant baculovirus to transiently transfect the HEK293 cells. 8-FcA can be used as a probe substrate in these studies. The results of this study confirm previous knowledge of the functioning of Ser112Ala, Met233Ile and Gly256Ala variants. More studies are needed about the effects of these variants on the transport of OATP1B3 drug substrates. Also studies about the location, cell membrane and total cell expression of the WTP variant are needed to evaluate reliably the reasons of its inactivity.
  • Rimpelä, Anna-Kaisa (2014)
    Certain drugs accumulate into pigmented tissues due to their binding to melanin, a macromolecule inside pigmented cells. Melanin can affect the drug's pharmacokinetics by acting as a drug reservoir. Binding can also cause toxic effects by accumulating compounds to pigmented cells. This thesis focuses on ocular melanin. The literature review covers the most common methods used in the study of ocular melanin binding and concentrates on in vitro methods and the analysis and usability of the results in pharmacokinetic modeling. The aim of the experimental part was to study melanin binding of a set of compounds in vitro with melanin isolated from the retinal pigment epithelium (RPE) and choroid of porcine eyes and with primary porcine RPE cells and then construct a pharmacokinetic model of melanin binding with STELLA® software and simulate it with the in vitro results. The compounds chosen for the study; nadolol, timolol, chloroquine, methotrexate, carboxydichlorofluorescein (CDCF) and dexamethasone, are small molecules with diverse physicochemical properties (octanol/water partitioning coefficient (logP), pKa, acid/base status). Some are also efflux substrates. The in vitro binding with melanin was studied at pH 7.4 and in addition at pH 5 for the acidic compounds, since the pH inside melanosomes where melanin is located is acidic. Porcine RPE cells were used to study the amount of uptake and rate of elimination of the set of compounds. The effect of efflux was also evaluated with a general efflux inhibitor probenecid. All the basic compounds bound to melanin in vitro. The acidic compounds did not seem to bind at pH 7.4 but bound at pH 5. Chloroquine, as expected, had the highest binding. In the cell studies, the uptake of chloroquine was significant, at least partly due to melanin binding. The other compounds were taken into the cells to a much smaller extent. The efflux inhibitor did not seem to affect the results. The results of the binding study were used in the models constructed of melanin binding and cellular pharmacokinetics. The constructed model was a very simple one not taking into account many factors affecting cellular pharmacokinetics. The results of both the in vitro studies and the model give a good idea of the importance of melanin binding in ocular drug delivery. The model can be used in the future as a base for more comprehensive models of the effect of melanin binding on ocular pharmacokinetics.
  • Laustio, Netta (2018)
    During the past few decades, the explosion of discovery in cancer and immunological research has led to the increased understanding of the interactions between the immune system and tumors. These developments have provided vital information about the immune system’s role in cancer development. It is evidenced that the immunity system is capable to distinguish tumor cells from normal tissue by recognizing tumor antigens that are exclusively expressed on tumor cells or are presented in greater amounts on tumor cells than normal cells. Consequently, the immune cells start to attack tumors for protecting the host. The possibility to use the immune system as a weapon against cancer cells leaded to the promising innovation – cancer immunotherapy – which aims to activate the body’s own immune system and its components to mount antitumor immune responses for eliminating cancer cells. The antitumor efficacy and high safety profile of several immunotherapeutic strategies have already been demonstrated thereby resulting in their integration into clinical practice. However, most patients have not benefited from cancer immunotherapy as a single treatment. In this regard, new innovative methods are clearly needed to overcome the obstacles hindering the clinical success of this field. Therapeutic cancer vaccines are emerging as attractive immunotherapies currently being evaluated in both pre-clinical and clinical studies. The purpose of cancer vaccines is to eradicate tumor cells by eliciting antitumor CD8+ T cell responses against the injected tumor antigens. Due to the ability to specifically kill tumor cells and simultaneously trigger immune responses against tumor antigens via direct oncolysis and by encoding transferred tumor antigens, oncolytic viruses are of significant interest for being used as in situ cancer vaccines. Despite these unique properties, several factors such as tumor immunosuppression and immune tolerance to targeted tumor antigens resembling antigens of normal tissues hamper the use of oncolytic vaccines in clinic. Instead of focusing only on CD8+ T cells, it has been suggested that giving more attention to CD4+ T helper cells, which are required for priming and expansion of CD8+ T cell responses, could be the key to improve the efficacy of cancer vaccines. Researchers have also demonstrated that an ongoing antigen-specific CD4+ T cell response can lead to the bystander activation of surrounding T cells with unrelated antigen specificities. Based on this theory, the hypothesis of this study was to employ the pre-existing immunological CD4+ memory against infectious pathogens in generating bystander CD8+ immunity against solid tumors. In this study, mice transplanted with poorly immunogenic B16-OVA tumors were pre-immunized with the chosen vaccine to induce immunological CD4+ memory against an infectious pathogen. Tumors were then treated with already developed cancer vaccine, which was peptide-coated conditionally replicating adenovirus (PeptiCRAd) complex. PeptiCRAd was constructed by electrostatically coating adenovirus with both pathogen-derived and tumor-derived peptide. The intratumorally injected double-coated PeptiCRAd complex was assumed to activate peptide-specific T cells and thus, result in anti-pathogen CD4+ T cell recall responses and the following bystander activation of antitumor CD8+ T cells, which can then mount an effective immune response to destroy cancer cells. The efficacy of this treatment was observed in pre-immunized mice by measuring the growth of injected tumors. The experiment was repeated identically with non-immunized naïve mice to see the difference in the results. The immunological background of this treatment approach was investigated by analyzing mouse tissue samples with standard immunological techniques including ELISA, IFN-γ ELISPOT and flow cytometry. This study showed that long-term immunological memory against the pathogen was successfully accomplished and the strongest inhibition of tumor growth in pre-immunized mice was achieved with double-coated PeptiCRAd, whereas the antitumor efficacy was not seen in naïve mice. Additionally, a new ex vivo method to detect pathogen-specific CD4+ T cells from spleen was developed and the stimulation of cell-mediated immunity by this treatment was supported by finding the highest levels of pathogen-specific CD4+ Th1 cells from mice treated with double-coated PeptiCRAd. Some encouraging results concerning the beneficial immune cell composition of tumors and tumor draining lymph nodes were also obtained from other performed experiments. Though further immunological analyses are required for understanding the precise mechanisms of action behind the treatment, the increased immunogenicity and antitumor efficacy of double-coated PeptiCRAd can still be considered as a consequence of the bystander effect, which can possibly be utilized for developing improved strategies to win the fight against cancer.
  • Koivunotko, Elle; Merivaara, Arto; Valkonen, Sami; Chinello, Lisa; Salmaso, Stefano; Korhonen, Ossi (2020)
    Biomimetic native nanofibrillated cellulose (NFC) hydrogel has recently proven its efficacy, safety and diversity at the site of pharmaceutical industry. Yet, properties for the long-term storage in dry condition at room temperature and feasible transportation needs to be developed for NFC hydrogel before it is suitable for freeze-dried biomedical applications. Our aim was to optimize freeze-drying cycle for NFC hydrogel formulation with suitable lyoprotective biomolecules and preserve its properties after freeze-drying process and reconstitution. NFC hydrogel formulations with different combinations of chosen biomolecules were freeze-dried, and physicochemical properties and rheological features were characterized. In addition, morphology of the freeze-dried cakes was studied. The effects of the biomolecules on the water contents in NFC systems were simulated for both of the crystal and amorphous ones. All the results of the characteristics were compared with the non-freeze-dried NFC hydrogel formulations. NFC hydrogel formulation, which had the most optimal preservation properties after freeze-drying and reconstitution, was optimized. We hypothesized that without any chemical modifications native NFC hydrogel can be successfully freeze-dried and subsequently reconstituted with the proper biomolecules only by using biological and natural materials, which are human and xenon-free for the further use in biomedical applications of the native NFC hydrogel.
  • Häkkinen, Aino (2016)
    There is a growing interest towards continuous manufacturing in pharmaceutical industry. When considering solid dosage form manufacturing and continuous wet granulation, twin screw granulation is the most studied technology. In spite of this, process knowledge is not as comprehensive as it is for batch granulation technologies such as high shear granulation. One problem with twin screw granulation seems to be bimodal and broad particle size distribution of granules. Specific causes of bimodal distribution and the ways to possibly gain unimodality either in granulation step or via dry milling are not fully understood. The purpose of this study was to optimize the ConsiGma25 continuous twin screw wet granulation process and study dry milling as a possible way of gaining unimodal particle size distribution in order to optimize particle size for tablet compression purposes. One aim was also to compress tablets from the obtained twin screw granules and evaluate the quality against high shear and direct compressed tablets of same formulation. Central composite circumscribed design was used as a design of experiments for twin screw wet granulation process. Factors (powder feed rate, screw speed, liquid to solid ratio (L/S ratio) and mill screen size) were varied in two levels. In total, 29 experiments were conducted. Tablet compression design was fractional as only 11 experiments from the twin screw granulation design were tableted. High shear granulation was conducted with Diosna P-1/6 using three different L/S ratios. All of these batches were compressed to tablets. Also direct compression was carried out. Formulation used had ibuprofen as active pharmaceutical ingredient, mannitol and MCC as fillers, HPC as binder and croscarmellose sodium as disintegrant. Lubricant, sodium stearyl fumarate, was blended to granules before tablet compression. Torque was measured during granulation in order to evaluate equilibration of the twin screw granulation process. It stabilized quickly and stayed stable after parameter changes and increased as L/S ratio and barrel fill rate increased. Particle size distribution and flowability of granules were analysed and tablets were analysed according to European and United States pharmacopoeias. Also tensile strengths and compactibilities were evaluated. Particle size distributions of unmilled twin screw granules were bimodal and broad. After dry milling, QicPic dynamic image analysis results showed unimodal particle size distributions for all experiments whereas Mastersizer laser diffraction analysis showed more unimodal distributions for experiments milled with smaller mill screen sizes. Mill screen size had the largest effect on particle size and as increased mill screen size increased particle size. Results showed that dry milling was a way to optimize particle size distribution for tablet compression purposes. Also flowability of formulation and process parameters needed to be optimized as granulation parameters had an effect on particle size and manufacturability was enhanced with better flowing formulation compared to previous study. Knowledge of the influence of L/S ratio, screw speed and powder feed rate, on granule size was gained. The effect of these process parameters varied depending whether d10, d50 or d90 was measured and particle size analysis method used. Increased L/S ratio and screw speed increased granule size as increased powder feed rate decreased it. Twin screw tablets showed higher tensile strengths and better compactibility compared to both high shear tablets and direct compressed tablets. Twin screw tablets showed also faster dissolution compared to direct compressed tablets probably due to of lower compression force. Mill screen size had the largest effect on dissolution properties of the twin screw tablets. When larger mill screen size was used, dissolution was slower due to larger particle size. Also increased powder feed rate made dissolution rate slower due to higher fill rate of the barrel. In other tablet properties analysed, no significant differences were seen between different twin screw experiments or between twin screw tablets and direct compressed tablets. All of the twin screw tablets and direct compressed tablets fulfilled the requirements of European and United states pharmacopoeia. As a conclusion, continuous granulation process was successfully optimized and high quality tablets resulted showing especially the effect of dry milling on granule size and shape as well as on tablet properties.
  • Vironen, Aleksi (2021)
    Evidence based medicines alongside with age-appropriate dosage forms are essential in enabling appropriate treatment for any patient group. Pediatric pharmacotherapy, however, is lacking age-appropriate dosage forms and research-based evidence regarding the dosing, efficacy, and safety of medicines. Orally administered drugs require manipulation to enable administration and are often used against the indications approved in the marketing authorization and summary of product characteristics (SmPC). This off-label use puts pediatric patients at risk for medicational errors and adverse drug reactions. The aim of this study was to investigate recent trends in oral dosage forms used in pediatric randomized controlled trials (RCTs), with emphasis on age appropriateness. The results could be utilized in developing evidence-based dosage forms, suitable for all pediatric patients aged 0-17 years, and manufacturable in a small scale in a hospital pharmacy. This study was conducted as a systematic review following the PRISMA Statement. The literature search was carried out from Pubmed and Scopus databases and it covered a five-year period of 2015-2020. References from the databases were entered to the Covidence systematic review platform. After removing duplicates 3333 articles were left for screening. Two independent researchers selected the articles first screening by title and abstract, and then by full text review. A qualitative content analysis was conducted on the included articles. Altogether 77 articles met the inclusion criteria. Dosage forms included were tablets (n=37), liquids (n=21), capsules (n=18) and multiparticulates (n=6). Majority of the dosage forms were conventional (n=49) compared to more advanced novel modified release and fixed-dose combination formulations (n=33). Based on our results, orally administered dosage forms used in the recent pediatric RCTs are still limited by poor acceptability, palatability, and the need to manipulate dosage forms to enable administration. These issues are similar to the ones related to the off-label use of medicine that compromise patient safety. Majority of the dosage forms included in our study were tablets, indicating a positive shift towards more safe and acceptable dosage form. Formulations were also evolving towards dispersible, extended-release and fixed-dose combinations that offer additional benefits for pediatric patients. The low number of children < 2 years old included in study populations and the poor acceptability profile reported by most studies limit our conclusions on an ideal age-appropriate dosage form. Further research is needed on unifying the guidelines used in pediatric drug development.
  • Jalonen, Daniel (2023)
    Orexin receptors have gained more attention due to increased knowledge of their physiological significance. The successful development of orexin receptor antagonists treating insomnia has enlarged the scope of research leading to an interest in developing small molecule agonists that have drug-like properties and induce activation in the orexinergic system. Transforming this idea into reality has remained an unaccomplished challenge. In this work, molecular mechanism of activation in orexin receptor type 2 is studied by conducting molecular dynamics simulations after capturing coordinates of active and inactive state crystal structures. The crystal structure coordinates define the starting pose for the protein and the ligand in the simulations. Preliminary steps prior to simulation include building the surrounding system and model building in which homology modeling is employed to reconstruct missing loops. A 100-nanosecond simulation is executed for both models. Active and inactive state models display stable binding event characteristics when examining the coordinate changes of every atom during the simulation. No major conformational changes are observed. The most congruent feature relative to the literature is the difference in the interactive role of residue Q3x32 between the simulations. The agonist forms two consistent hydrogen bonds with the upward-shifted Q3x32 while an inactive downward-facing version is observed in the antagonist model. Some residues present unexpected features omitting comparative functions of literature, which along with general inconsistency of protein-ligand contacts undermine the reliability of models heavily. The simulations conducted need to be extended to produce a hypothesis for the activation mechanism because of the defects around simulation protocols and the low quantity of simulation runs.
  • Miettinen, Ilkka (2016)
    Multi-drug tolerance is a phenomenon, in which microorganisms normally susceptible to an antimicrobial agent are able to withstand a treatment via phenotypic alteration. The tolerance is conveyed by a microbial subpopulation that is in a non-replicative and metabolically inactive state also known as persistence. Through this kind of dormancy, the subpopulation may survive an otherwise appropriate course of antimicrobials, since the majority of the drugs target cellular division or metabolism. Upon the reduction of the surrounding antimicrobial concentration the multi-drug tolerant cells - persisters - become resuscitated thus allowing repopulation. As opposed to the more widely acknowledged challenge of antimicrobial resistance, the offspring of the specialist survivor cells are genetically identical to the susceptible majority. Persisters are especially abundant in biofilms, a microbial lifestyle characterized by aggregated microcolonies that are covered in a self-produced slimy matrix known as extracellular polymeric substance (EPS). Partly owning to this protective matrix, biofilms are inherently somewhat tolerant to antimicrobial chemotherapy. Moreover, microbes confined in a biofilm are additionally protected against the components of the host immune system. Conversely, it is assumed that persisters in planktonic, i.e. freely floating state, are easily cleared out by white blood cells. Combined, the immune evasive properties of biofilms and the remarkable multi-drug tolerance of persisters give rise to recalcitrant infections that are immensely difficult to eradicate. The described phenomenon constitutes crucially to the major healthcare challenge of chronic, treatment-resistant infections. Tuberculosis, cystic fibrosis lung disorder, bacterial endocarditis and infections related to indwelling medical devices are only a few examples of such problems. Despite the need for antimicrobials with anti-persister efficacy, no such therapeutics is available and very few are being investigated - one important factor being the lack of relevant drug discovery platforms. Therefore, the aim of this study was to develop an anti-persister assay and to carry out a pilot screening of natural product derived bioactive compounds. Based on the notion that persisters are enriched in bacterial cultures that have reached the stationary phase of growth, a persister model was designed using Staphylococcus aureus ATCC 25923 as the test strain. The bacteria were grown in liquid cultures until they reached the stationary phase and subsequent experimentation was carried out to confirm the tolerant state. After the stationary phase persister model was validated, a small pilot screening of natural products was undertaken in the hope of finding novel anti-persister activity. Mitomycin C, a cytotoxic drug with an existing anti-cancer indication was assigned as the positive control compound because of its previously established anti-persister activity. Since it is common for all of the persister-related diseases that the target microorganisms reside within a protective biofilm, an additional assay based on biofilm regrowth was designed to characterize the hit compounds on a more clinically relevant platform. The persister model culture was shown to be tolerant to conventional antibiotics. The re-induction of metabolic activity by diluting into fresh medium recovered the antimicrobial susceptibility expectedly. A total of 4 compounds were identified as anti-persister hits in the pilot screening campaign. Chromomycin A3, dehydroabietic acid, mithramycin A and oleanolic acid were all able to reduce the viable bacterial count in the stationary phase persister model more than 2 logarithmic units at 100 µM. Mithramycin A was the most potent, reducing the viability over 6 log units. The model compound mitomycin C reduced the viable counts 5.49 (± 0.96) logarithmic units. Out of the 4 hits, dehydroabietic acid was selected for the biofilm relapse assay because of its favourable biocompatibility properties. It reduced regrowth for the treated biofilms by 4 logarithmic.
  • Toivonen, Johanna (2012)
    Parkinson's disease is a neurodegenerative disorder where dopaminergic neurons in substantia nigra are gradually destroyed. Less than 10% of Parkinson's disease cases are genetic. For example mutations in α-synuclein, LRRK2-, parkin-, PINK1- and DJ-1 are known to cause Parkinson's disease. There is still no curative treatment for Parkinson's disease. Alpha-synuclein is linked to Parkinson's disease through Lewy bodies. Three point mutations causing Parkinson's disease have been found in a gene coding α-synuclein. Alpha-synuclein has been expressed in Drosophila melanogaster, C. elegans and mouse. Main function of LRRK2-protein is thought to be kinase activity. Mutations in LRRK2-gene are the most common known cause of Parkinson's disease. LRRK has been expressed in Drosophila melanogaster, C. elegans and mouse. LRRK2 knock-out Drosophila melanogaster and mouse have also been studied. Parkin is a neuroprotective protein and its deficiency results in a loss of neurons in substantia nigra. Mutations in Parkin cause 50% of recessive Parkinson's disease. Parkin knock-out Drosophila melanogaster and mouse and Drosophila melanogaster and mouse expressing human Parkin are Parkin animal models. PINK1 is a mitochondrial protein coded by nucleus. DJ-1 is thought to have a part in mitochondria maintenance and protection. Both PINK1- and DJ-1 knock-out Drosophila melanogaster and mouse have been studied. None of the genetic animal models of Parkinson's disease is identical to symptoms and pathology of human Parkinson's disease. The purpose of the experimental part of this thesis was to examine non-drug induced behavioural test and Cerebral dopamine neurotrophic factor (CDNF) in 6-OHDA lesioned rats. CDNF protects and restores dopaminergic neurons. The non-drug induced behavioural tests included in this study were stepping test, cylinder test and staircase test. An old and widely used drug induced test for Parkinson's disease, amphetamine-induced rotation test, has problems that have led to a seek for replacing and complementary test methods. In amphetamine-induced rotation test dopamine agonist is given to a unilaterally 6-OHDA lesioned animal. The agonist causes rotational behaviour that can be measured with designed equipment. The stepping test measures forelimb akinesia in rats. In the experimental setting the rat is moved sideways when it is held only one front paw on a table and adjusting steps are counted. In the cylinder test front paw preference is measured. In the experimental setting the rat is placed in a transparent cylinder and the front paw preference is counted on rears and on ground contacts after a rear. The staircase test measures front paw coordination and function. In the experimental setting the number of sucrose pellets picked up from a double staircase is counted. There were no significant differences between lesioned groups in stepping test, cylinder test or in staircase test. It is possible that the 6-OHDA lesion used in the experiment was not extensive enough. Different non-drug induced behavioural tests supplement each other and they should be combined for the best result. Combining different behavioural tests enables more reliable results and versatile information than the amphetamine-induced rotation test alone.
  • Koljonen, Petri (2012)
    Parkinson's disease is characterized primarily as a bradykinetic disorder with severe nigral cell loss. In addition to motor symptoms, up to 85 % of patients with Parkinson's disease experience pain and in about 60 % of cases pain is related to Parkinson's disease. Most of it is classified as musculoskeletal pain. Bradykinesia and muscle cramps lead to pain by causing malpositions of joints and trunk. Up to 40 % of parkinson patients experience pain caused by dystonia. Neuritic or radicular pain is also related to Parkinson's disease. Less than 10 % of patients have primary central pain. Pain threshold and nociceptive flexon reflex threshold are lower among patients with Parkinson's disease than in healthy subjects. Common comorbidities, namely restless legs syndrome and depression can also exacerbate pain. The pathology of pain in patients is not well understood. It is known that basal ganglia take part in pain perception and modulation. Lesions in basal ganglia can interfere pain perception and cause the exacerbation of pain. The modulation of pain in central nervous system is altered and descending inhibitory tracts are thought to work insufficiently. Levodopa alleviates the pain in about 60 % of patients with Parkinson's disease suffering from pain. Levodopa normalizes dopamine function at least partly in basal ganglia and that way alleviates the pain caused by dysfunction of dopamine tracts. Levodopa relieves motor symptoms and so alleviates the secondary pain caused by muscle cramps and stiffness. Levodopa raises the pain thresholds of patients to normal level. Levodopa may have also a direct analgesic effect via dopamine D2 receptor activation. The mutations of the gene that codes catechol-O-methyltransferase (COMT) change its activity and are related to pain perception. Low COMT activity is related to several functional differences including increased sensitivity to pain and increased response to opioids. Also COMT inhibitors sensitize mice and rats to pain. The mechanism underlying the sensitization is not well understood. We examined the effects of COMT gene disruption and COMT inhibition in acute pain models. In the first part of our study, we examined the effect of COMT inhibitor OR-486 in COMT deficient mice. We tried to clarify wether sensitization to pain is caused by COMT inhibition or some other mechanism. We also tested the effects of endogenous opioids (swim stress) and exogenous opioid (morphine) in COMT deficient mice. In the second part, we tested the effects of an atypical COMT inhibitor CGP 28014 in acute pain models. CGP 28014 does not inhibit COMT in vitro but it inhibits the Omethylation of catecholamines in vivo. The main finding of our study was the sensitization to pain caused by CGP 28014. The result gives support to hypothesis claiming that sensitization to pain is caused by O-methylation of catecholamines. The results of our study are also in line with the theory that low COMT activity is related to pain sensitization and increased response to opioids.
  • Uhari, Johanna (2010)
    Part I: Parkinson's disease is a slowly progressive neurodegenerative disorder in which particularly the dopaminergic neurons of the substantia nigra pars compacta degenerate and die. Current conventional treatment is based on restraining symptoms but it has no effect on the progression of the disease. Gene therapy research has focused on the possibility of restoring the lost brain function by at least two means: substitution of critical enzymes needed for the synthesis of dopamine and slowing down the progression of the disease by supporting the functions of the remaining nigral dopaminergic neurons by neurotrophic factors. The striatal levels of enzymes such as tyrosine hydroxylase, dopadecarboxylase and GTP-CH1 are decreased as the disease progresses. By replacing one or all of the enzymes, dopamine levels in the striatum may be restored to normal and behavioral impairments caused by the disease may be ameliorated especially in the later stages of the disease. The neurotrophic factors glial cell derived neurotrophic factor (GDNF) and neurturin have shown to protect and restore functions of dopaminergic cell somas and terminals as well as improve behavior in animal lesion models. This therapy may be best suited at the early stages of the disease when there are more dopaminergic neurons for neurotrophic factors to reach. Viral vector-mediated gene transfer provides a tool to deliver proteins with complex structures into specific brain locations and provides long-term protein over-expression. Part II: The aim of our study was to investigate the effects of two orally dosed COMT inhibitors entacapone (10 and 30 mg/kg) and tolcapone (10 and 30 mg/kg) with a subsequent administration of a peripheral dopadecarboxylase inhibitor carbidopa (30 mg/kg) and L- dopa (30 mg/kg) on dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens of freely moving rats using dual-probe in vivo microdialysis. Earlier similarly designed studies have only been conducted in the dorsal striatum. We also confirmed the result of earlier ex vivo studies regarding the effects of intraperitoneally dosed tolcapone (30 mg/kg) and entacapone (30 mg/kg) on striatal and hepatic COMT activity. The results obtained from the dorsal striatum were generally in line with earlier studies, where tolcapone tended to increase dopamine and DOPAC levels and decrease HVA levels. Entacapone tended to keep striatal dopamine and HVA levels elevated longer than in controls and also tended to elevate the levels of DOPAC. Surprisingly in the nucleus accumbens, dopamine levels after either dose of entacapone or tolcapone were not elevated. Accumbal DOPAC levels, especially in the tolcapone 30 mg/kg group, were elevated nearly to the same extent as measured in the dorsal striatum. Entacapone 10 mg/kg elevated accumbal HVA levels more than the dose of 30 mg/kg and the effect was more pronounced in the nucleus accumbens than in the dorsal striatum. This suggests that entacapone 30 mg/kg has minor central effects. Also our ex vivo study results obtained from the dorsal striatum suggest that entacapone 30 mg/kg has minor and transient central effects, even though central HVA levels were not suppressed below those of the control group in either brain area in the microdialysis study. Both entacapone and tolcapone suppressed hepatic COMT activity more than striatal COMT activity. Tolcapone was more effective than entacapone in the dorsal striatum. The differences between dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens may be due to different properties of the two brain areas.
  • Helminen, Heidi (2017)
    Obesity is considered one of the major public health challenges. One way to control obesity is to regulate appetite. Because brain is the primary regulative unit responsible for food intake, the research in this field has now been allocated especially to the central nervous system. The aim of this thesis was to clarify the role of cholinergic projections from pedunculopontine tegmentum (PPT) to lateral hypothalamus (LH) in food intake. In this study, DREADD-technology (designed receptors exclusively activated by designer drugs) based on chemogenetics was utilized with a gene manipulated mouse strain. For the experimental part of this work the mice were divided in three separate groups: one transducted with an activating DREADD-receptor (hM3Dq), one transducted with an inhibiting DREADD-receptor (hM4Di) and one transducted only with a fluorescent protein called m-Cherry. The last group was also defined as a control group of this study. In addition, the gene which coded m-Cherry fluorescent protein was transducted together with hM3Dq- and hM4Di-receptor genes for the first two groups to be able to examine the receptor expression later. At the baseline level, no differences were observed in food intake between the three study groups. The food intake did not differ between the groups while clozapine-N-oxide (CNO), a selective DREADD-receptor ligand, was administered straight into the LH area (0,03 µg/injection) with or without fasting of the animals. While administrating CNO to the mice intraperitoneally (1 mg/kg), the hM3Dq-group mice were observed to consume more food compared to the hM4Di-group or the control group. This difference was detected while food consumption was examined cumulatively during total four measuring hours. When the animals were fasted before the intraperitoneal administration test, however no differences were found between the study groups regarding food intake. As a conclusion of this study, cholinergic projections from pedunculopontine tegmentum (PPT) to lateral hypothalamus (LH) were not regulating food intake in mice. However, the cholinergic cells in PPT and some of their axons might be involved in the regulation of food intake while the food consumption is studied continuously and long-term. More studies are required to better define the role of the cholinergic projections from pedunculopontine tegmentum (PPT) to lateral hypothalamus (LH) in food intake.
  • Honkasalo, Oona (2018)
    Cancer immunotherapies aim to target the immune defence mechanisms of the body specifically and efficiently against the tumour tissue. Cancer vaccines and oncolytic viruses are forms of active immunotherapies, which require patients having a properly functioning immune system. The vaccines are based on the administration of tumour antigens into the body to which the immune system reacts. However, often the response is not robust enough. The oncolytic viruses in turn kill the cancer cells which causes the release of antigens from the tumour tissue. Viruses usually elicit a strong immune response but sometimes it is targeted too much against the virus instead of the tumour. Oncolytic vaccine is a composition of an oncolytic virus and a cancer vaccine. Tumour antigens can be coded to the genome of the virus therefore, when the virus invades tumour cells they start to produce the antigens. Eventually the cancer cells are also destroyed due to viral replication. The antigens can be tumour-associated that is, they are expressed in healthy tissues too. Their usage is not always efficient which is why an interest towards utilizing tumour-specific antigens has been increased. Considering the expression of antigens, tumour tissue is very heterogenous and distinctive between patients. Hence, utilizing mutated patient unique neoantigens would enable the development of personalized tumour-specific oncolytic vaccines. Genetic modification of viruses is complicated thus, an easier way to insert the neoantigens to the virus has been invented. The developed oncolytic vaccine platform is called PeptiENV, and it is designed to use with enveloped viruses. The idea is to fuse tumour-specific antigens onto the envelope of the virus and eliminate the need of gene insertion. The aim of this study is to investigate in vivo the efficacy of PeptiENV in preventing tumour growth and eliciting a tumour-specific immune response. An object is also to observe survival times of the treated animals. Furthermore, the preservation of infectivity is studied in vitro. The research was executed with two potential oncolytic viruses, vaccinia virus (VACV) and herpes simplex virus type 1 (HSV-1). The PeptiENV complex was formed by using an artificial tumour antigen, ovalbumin epitope SIINFEKL, which was attached to the viral envelope with cell penetrating peptide (CPP) or cholesterol anchor. The preservation of infectivity was examined by measuring cell viability of PeptiENV infected cells. Animal experiments instead were performed with a mouse melanoma model created with B16-OVA cells, which express ovalbumin and therefore the antigen epitope SIINFEKL. PeptiENV was compared to control treatments which were virus, SIINFEKL peptide and complexation medium only. Treatments were administered as intratumoural injections. Tumour growth was followed by measuring the size of implanted tumours every other day. With flow cytometry, tumour-specific immune response was assessed by acquiring the relative amount of SIINFEKL-specific CD8+ T cells in the tumour tissue. Euthanizing dates were registered in order to observe the survival of the mice. According to the in vitro results, conjugation of peptides to the virus does not affect infectivity. In addition, the in vivo studies show that PeptiENV VACV CPP prevents tumour growth the most. Difference in tumour growth between PeptiENV VACV CPP and control treatments is significant. Mice injected with the same treatment also lived considerably longer than mice injected with virus, peptide or medium only. Also, PeptiENV HSV-1 hinders tumour growth distinctly more than virus only and slightly more than SIINFEKL only, but unfortunately it did not have an evident impact on the survival time. In both experiments, the PeptiENV treatment elicits the largest proportional amount of SIINFEKL-specific CD8+ T cells. In other words, PeptiENV engenders a tumour-specific immune response. In the PeptiENV VACV study the difference to control treatments is clearer than in the PeptiENV HSV-1 study. At present, the PeptiENV platforms performs better with VACV than HSV-1. With further investigations however, the results can be verified and improved. All in all, the results are encouraging. The PeptiENV platform shows great promise for being a part of personalized cancer immunotherapy developments in the future.
  • Luoma, Maaria (2018)
    Inappropriate polypharmacy refers to a situation where more than appropriate amount of medicines are used by a patient. Aged people with multiple morbidities and medications use a lot of health care services and are thus especially vulnerable to iatrogenesis, the health hazards resulting from the acts of a health care system. As a part of normal ageing, geriatric syndromes (e.g. falls, delirium and urinary incontinence) are clinical conditions and symptoms crossing several organ systems and they cannot be connected to a certain individual disease. Geriatric syndromes complicate recognition of adverse drug reactions on aged. This increases the risk of prescribing cascade, where medicines are prescribed to treat adverse drug reactions caused by another medicine. In this master´s thesis the root causes for inappropriate polypharmacy and drug-related problems (DRP) with home-dwelling aged were researched retrospectively from the viewpoint of risk management. Research method was based on root cause analysis (RCA) that was simplified suitable for this research. Research material was based on an intervention research conducted in 2015– 2017 on home-dwelling aged receiving regular home care from the City of Lohja, Finland. In the intervention research, a coordinated community-based medication management model for home-dwelling aged in primary care was developed to identify homedwelling aged with clinically significant drug-related problems. As research material, there were five (n=5) patient cases used who received comprehensive medication review (CMR) in the intervention research to solve their drug-related problems. The research material composed of individual patient interviews conducted at patients’ homes as a part of their CMR visits. Also, the nurses (n=3) of home care and physicians (n=2) from local health centres having participated in the treatment of the home-dwelling aged in question, were interviewed individually. Markings made in the patient records were utilized as well as research material. The interviews of the nurses and physicians were recorded, transcribed and analysed with inductive content analysis considering principles of root cause analysis. According to the nurses and physicians, central clinically significant medication-related problems with home-dwelling aged are various prescribing care parties, multiple medications, the increased use of over-the-counter (OTC) medicines and natural products, the uncertainty of health care professionals of the medication of a home-dwelling aged as well as the occurrence and medication of pain and sleeping disorders with aged. Other essential problems related to the health care system are various attending physicians, obscurely recorded medication data in patient record system, the use of benzodiazepines and other psychopharmaceuticals and ignored renal function in medicine dose adjustment. Problems related to home-dwelling aged are attachment for medicines, resistance to change and desire to take care of their own medication. In addition, memory disorders and vertigo were mentioned as problems related to the medication of aged. Seven root causes for inappropriate polypharmacy and drug-related problems were observed: lack of health care resources, segmented treatment between various health care parties, varying skills and knowledge of health care professionals, ambiguous division of responsibilities between health care professionals, challenges in communication between different care parties, the heterogeneity of patient record systems and problems related to their use as well as the knowledge, opinions and personal situation of a home-dwelling aged. Based on the research, the medication of home-dwelling aged should be improved by striving for centralizing care in one physician either on private or public health care. Among home care nursing personnel there is a need for additional training on medications and pharmacists should participate in regular medication reviews for home-dwelling aged. Patient record systems and data transmission between them should be improved and medication data should be recorded more precisely. Cooperation and communication between home care and health centre should be developed and the division of responsibilities should be clarified. Participation of the home-dwelling aged and their relatives in the care should be promoted. Furthermore, geriatric expertise should be utilized better in the care of the home-dwelling aged.
  • Pottonen, Riitta-Liisa (2015)
    Medication safety is safety related to the use of medicines. Medication errors are drug treatment related events which can lead to medication safety incidents. Medication process is multi-professional teamwork which contains a risk of medication error on every step. It is important to identify potential safety risks in order to prevent the risk events. Medication errors can occur for example during the transfer of the prescription information in to the medication list. It is important that medication lists are accurate and up to date so that patient's medication therapy is optimal. The aim of this study was to assess whether the primary health care medication lists are up to date, accurate and easy to read. The aim was also to identify what kind of information in medication lists was open to interpretation. In one of the municipalities, the medication lists at home were compared to the medication lists at primary care to see whether both lists had identical information on the medication. The data of this study consisted of 240 medication lists from primary care units in three Finnish municipalities. The lists contained altogether 3062 medications. Most of the lists were printed from the patient information systems. Some of the lists were copied from the home medication lists. All medication lists were systematically reviewed and issues open to interpretation were documented in a structured Microsoft Excel table. The data were transferred to SPSS 20 Program for statistical analysis. Most (73%, n=174) of the medication lists (n=240) were incomplete. One-fifth (n=612) of the medications in use (n=3062) contained missing information on medicines. The total number of discrepancies was 807 (mean 3.4 discrepancies per medication list). The most common discrepancies were related to the time of administration (n=277) and dosages (n=241). Duplicate medications included a lot of confusing information. Discontinued medications were not always clearly marked. In only one of the municipalities the medication lists had a space for marking the indication. There were some differences between home medication lists (n=62) and primary care medication lists. ™ Based on this study medication lists have a lot of discrepancies and ambiguities in their information content. The medication lists do not always accurately tell the patient's current medication. Interpretation of inaccurate medication lists consumes unnecessarily doctors' and nursing staff's time. Inaccurate medication lists are a risk to patient safety. It is also important that the medication lists would be similar in all health care units. Electronic prescriptions, the National Health Archive and medication list developed by Information Management Service of Healthcare are expected to solve at least some of the problems related to medication lists.
  • Suvanto, Satu (2014)
    P-glycoprotein is an ATP-dependent efflux protein expressed in many tissues which participate in absorption, distribution and elimination of drug molecules. It can mediate clinically significant drug-drug interactions. Characteristics of P-gp have been studied widely and crystal structure of mouse P-gp has been successfully determined. P-gp binds its substrates either directly from cell membrane or from cytosol and it has at least three separate binding sites. P-gp has wide selection of substrates from many therapeutical groups. According to the latest computational models, a typical P-gp substrate can be defined with the help of molecule structural factors rather than physicochemical properties. However function of P-gp is very complex which is why drug-drug interactions should be studied for each drug pair separately. In addition expression of P-gp is regulated by nuclear receptors PXR and CAR thus P-gp induction is separate, which also complicates P-gp mediated interactions. P-gp substrates celiprolol, talinolol, aliskiren and fexofenadine have in vivo interactions with P-gp inhibitors or inducers. The objective the experimental work was to study suitability of two in vitro methods, MDCKII-cell permeability assay and MDR1-vesicle transport assay, for predicting in vivo effect of drug-drug interaction. ATP-dependent transport of substrates was determined in membrane vesicles extracted from human P-gp expressing Sf9 cells. Cell assay was used to determine efflux ratio (ER) for all the substrates alone and efflux ratio with P-gp inhibitor itraconazole for the substrates which have reported in vivo interaction with itraconazole. All compounds showed ATP dependent transport in MDR1-vesicles and celiprolol, talinolol and fexofenadine showed ER over 1 in MDCKII-MDR1 cells thus according to vesicle assay and ER-value they are P-gp substrates. However ER of talinolol and fexofenadine was not affected by inhibitor itraconazole, thus the drugs did not fulfil the inhibition criteria of FDA for P-gp substrates. The performing of interaction test was possible failed due lack of pre-incubation of the cells with the inhibitor. Talinolol had the highest ER in thus according to cell experiments talinolol has P-gp dependent transport. Aliskiren ER was not obtained because of the low recovery of the drug but it had clear ATP-dependent transport in the vesicle assay as was expected according to in vivo results. According to in vitro results and in vivo studies celiprolol is a poor P-gp substrate whereas fexofenadine showed P-gp mediated transport both in vitro and in vivo. The results suggest that significance of drug interaction is difficult to predict with the vesicle and the cell assay but they can be used to recognize P-gp substrates.
  • Hurmalainen, Virpi (2021)
    P-glycoprotein is an efflux transporter of the ABC family. It is expressed mainly in tissues that have a role in limiting the absorption and distribution of xenobiotics in the body or their elimination. P-glycoprotein is known to have an important role for example in the blood-brain barrier and in protecting the fetus from xenobiotics in the mother’s blood stream. Genetic polymorphisms in transporter proteins can cause individual differences in the pharmacokinetics of drug substances, which can lead to differences in drug efficacy or side effects. In the ABCB1 gene, which codes for p-glycoprotein, several polymorphisms have been discovered. The frequencies of these polymorphisms vary in different ethnic populations. Previous studies have shown that the effects of these polymorphisms are often substrate-dependent. Since there are several confounding factors usually present in clinical association studies, in vitro studies are needed to clarify the effects of individual polymorphisms. Polymorphisms can be studied in vitro by making intentional mutations to the gene sequence and expressing the variant gene in a suitable cell line. In this study four variant p-glycoprotein genes (c.781A>G, c.1199G>T, c.2005C>T and c.3421T>A) were created by site-directed mutagenesis, and expressed in HEK293 cells using a baculovirus recombinant protein expression method. The effects of the polymorphisms were studied by determining the expression level and the transport acitivity of the variant proteins compared to the wild-type. Western blot was used to determine the expression level and a calcein accumulation assay in HEK293 cells was used to compare the transport activities. Also a membrane vesicle transport assay with n-methyl quinidine was set up and optimized, but the variants were not yet studied with this method during this study. In this study no statistically significant differences were found in the transport activities of any of the four variants compared to the wild-type p-glycoprotein. Also the differences in protein expression level between wild-type and variant proteins were small. However, because of the previously reported substrate dependency of polymorphism effects, it would be beneficial to study the variants with at least one other substrate and one other assay method, and thus the membrane vesicle transport assay would be useful to further compare the transport activities of variant proteins to the wild-type p-glycoprotein.
  • Urpelainen, Katja (2021)
    Cardiomyocyte oxygen deprivation followed by apoptosis and cardiomyocytes being replaced with fibrotic tissue can lead to heart failure. Cardiovascular diseases are the most common cause of death world-wide, contributing to 17.8 million deaths in 2017. Treatments currently available aim to maintain cardiac function but are unable to repair the damage, resulting in a poor prognosis for heart failure. Cardiomyocytes are able to proliferate but the endogenous mechanisms of cardiac repair are insufficient to replace the damaged cardiomyocytes, as only an estimated 0.3-1 % of adult cardiomyocytes are regenerated annually. It is known that before birth and up to seven days after birth mice can maintain ability to regenerate cardiomyocytes even after large damage, but this capability is lost within seven days following birth. After this, cardiomyocytes exit cell cycle and will not re-enter it sufficiently to enable cardiac repair. In adults the growth of heart muscle results mainly from hypertrophic growth meaning that the cells grow in width and length. Cardiomyocyte regeneration is an important therapeutic target to which there are no effective pharmacological therapies available yet. The aim of this study was to investigate the effect of 14 novel compounds on cardiomyocyte viability, phenotype and cell cycle activation. Novel compounds were synthesized at the Faculty of Pharmacy, Division of Pharmaceutical Chemistry and Technology, University of Helsinki in Finland. Initial toxicity and cell viability screening was conducted with lactate dehydrogenase assay (LDH assay) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay) using COS-1 cells. Based on these assays tolerable concentrations of compounds were determined. Activity analysis was conducted using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) and immunocytochemistry staining in conjunction with high-content analysis (HCA). Stress response was measured by imaging and analyzing expression of pro-B-type natriuretic peptide (proBNP) and cell cycle activation was imaged and analyzed by using 5- bromo-2’-deoxyuridine (BrdU) as a marker of active cell cycle. In addition, the DNA content of the cardiomyocytes was measured using 4’,6-diamidino-2-phenylindole (DAPI) staining as well as cardiomyocyte morphology investigated with cardiac troponin T (cTnT) staining. One of the compounds, K6, decreased proBNP expression, which can be considered as a sign of decreased stress response. However, K6 also decreased the number of BrdU positive cardiomyocytes that can be considered as a sign of decreased cell cycle activity. Together these markers indicate that the decreased activity may not be due to a stress response caused by the compound. Another compound, K12, increased proBNP expression in all tested concentrations and it also decreased the number of BrdU positive cardiomyocytes. Together these could be considered as an indication of cardiotoxicity. The rest of the compounds did not exhibit remarkable biological activity or there was too great variance between the results of the independent experiments (n=3) to draw definite conclusions. Compounds for this study were chosen for the sole reason of not been tested for biological effects before. Using a defined compound library or screening a larger number of compounds could deliver more predictable results. Early toxicity and viability screenings were a good approach allowing to define toxic compounds and concentrations before continuing with further studies. Pharmacological therapies to induce cardiac regeneration will continue to be an important area of interest in cardiovascular drug research. Phenotypic screening in conjunction with high-content analysis offers variable and statistically significant data on cardiomyocyte proliferation and stress response. The results of the screening could be improved with careful selections of test molecules based on their structure and biological activity. Early toxicity and viability screening further improve the predictability of results. As a result of this study a compound that would induce cardiomyocyte proliferation was not found, however, one compound that seemed to decrease cardiomyocyte stress response was detected and this compound could be of interest for further studies.
  • Tseloev, Idris (2018)
    An ultra-high-pressure liquid chromatography method was used for simultaneous detection of 25 small peptide hormones and their metabolites in urine after solid-phase extraction. This method is first screening step in anti-doping analysis of urine samples. It should be fast, generic and able to detect any sample that may contain a prohibited substance while avoiding false negatives and reducing false positive results. Detection was achieved using quadrupole time-of-flight mass spectrometry coupled with electrospray ionization source in positive mode. Analytes included growth hormone secretagogues, gonadotropin releasing factors, anti-diuretic hormones and their metabolites which are all covered by the list of prohibited substances of the World Anti-Doping Agency (WADA). The practical part of investigation was done in United Medix Laboratories and the aim of study was to expand current screening method by adding new compounds. Optimal experimental conditions were stablished after investigation of different parameters concerning sample preparation and instrumental analysis. The extraction procedure was done by using weak cation exchange SPE with two washing steps (Milli-Q water and methanol), and elution with 5 % formic acid in methanol. The procedure was validated in terms of recovery, specificity, limits of detection, stability and robustness. Recovery was evaluated with 10 ng/ml concentration of analytes and the rest of validation procedures were done at half of minimum required performance level set by WADA (1 ng/ml). Recoveries ranged from 2,6 to 85 % with LODs from 0,01 to 1,76 ng/ml. The suitability of the method was assessed by analyzing different spiked urine specimens containing target substances.