Browsing by master's degree program "Master's Programme in Pharmacy"
Now showing items 21-40 of 133
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(2024)Alzheimer's disease is a progressive neurodegenerative disease of the central nervous system, in which accumulation of amyloid β plaques and hyperphosphorylated tau proteins cause neuronal death, loss of synapses, and impaired neurotransmission. Alzheimer's disease's main symptoms are memory dysfunction (dementia) and decline in cognition. One of the most significant factors believed to cause dementia in Alzheimer's disease is the destruction of dendritic spines. Dendritic spines are small protrusions of dendrites where most of the glutamatergic synapses are located and where mainly excitatory synaptic functions occur. Loss of dendritic spines is directly correlated to the loss of synaptic function, which then causes memory dysfunction and impaired cognition. Also, the morphology of dendritic spines is important for their stability and strength. The spine head's size is correlated to the number of postsynaptic receptors. Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are unconventional neurotrophic factors, which have neuroprotective and -restorative effects via regulating endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Accumulation of unfolded and/or misfolded proteins in the lumen of ER causes ER stress, which then activates UPR, which again maintains protein homeostasis by reducing the amount of unfolded/misfolded proteins. In this study, we injected CDNF, MANF or PBS into the hippocampus of C57BL/6 mice to analyze whether they increase dendritic spine density and improve their morphology, especially by increasing the density of mature spines. This study shows that MANF increases dendritic spine density and CDNF does not. MANF increases the density of most of the mature and immature spines but interestingly decreases the density of mature stubby spines. These results are very promising and MANF's effect on dendritic spine formation should be studied further. In another separate study, we investigated whether CDNF and MANF activate the ERK1/2 pathway on organotypic hippocampal slices. This pathway is important for memory formation, and another neurotrophic factor, brain-derived neurotrophic factor (BDNF), has been shown to increase dendritic spine density by stimulating the ERK1/2 pathway. CDNF and MANF activate UPR, but there is some evidence that UPR might also activate ERK1/2, which could then explain CDNF's and MANF's mechanism of action. CDNF and MANF-treated hippocampal slices had visually thicker and darker bands in ERK1/2 antibody staining but any conclusion cannot be made because of the sample sizes being too small.
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(2024)Cardiac fibrosis (CF) is a physiological response to various stress factors encountered by the heart, with the aim of maintaining proper functioning of this vital pump in an altered situation such as increased mechanical stress or sudden injury in heart muscle. CF is characterized by excessive production of extracellular matrix (ECM) components and stress fibers in cardiac tissue, accompanied by morphological changes of the heart muscle. The responsible cells behind these changes are fibroblasts (FBs) that undergo phenotypic change by transdifferentiating into myofibroblasts (myoFBs). Although being initially a supportive response, CF can lead to deterioration of the heart performance and even heart failure, if prolonged. Given the lack of effective enough therapies against CF, and the strong involvement of CF in cardiovascular diseases (CVDs) that are associated with high mortality rate, the need for new effective therapies is urgent. Indeed, a diversity of approaches to fight CF have been proposed, among them protein kinase C (PKC) and its signaling cascades. PKC has been shown to play a role in fibrosis and many studies suggest antifibrotic properties of PKC, yet the results are challenged by the opposite findings. Despite the dichotomous results, new small molecules that function as partial agonist of PKC seemed to be a promising strategy for the treatment of fibrosis. To further explore the role of PKC activation in CF, the aim of this study was to first develop and characterize a human cardiac fibroblast (HCF)-based CF model, in which the effects of seven new PKC modulators on HCFs could then be evaluated. To create the CF model and provoke a fibrotic response, HCFs were treated with either transforming growth factor β1 (TGF-β1), Angiotensin II (Ang-II), endothelin-1 (ET-1), or combination of treatments, followed by determination of HCF proliferation activity and α-smooth muscle actin expression (α-SMA), a marker of myoFBs. After the treatments, the original goal was to continue in compound testing phase by exposing the HCFs to the PKC-modulators to see whether any differences could be determined in α-SMA expression or proliferation activity. However, no considerable effects of fibrosis-inducing treatments on the activation of HCFs were observed, thus preventing this progression. Nevertheless, toxicity tests were performed on the compounds and the results indicated relatively low overall toxicity for the lower concentration: six out of seven compounds yielded over 70% HCF viability at 3 μM concentration with three of them reaching even over 80% viability, while the corresponding value for the previously published PKC agonist HMI-1a3 was 54%. Although these results are promising for the lower concentrations of PKC-modulators, it is obvious that more in-depth studies are required prior to drawing any unambiguous conclusions.
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(2021)Huntington’s disease (HD) is a rare but devastating neurodegenerative disease, progressively culminating in severe brain atrophy and death. The disease is caused by an inherited mutation resulting in a CAG trinucleotide repeat expansion in the huntingtin gene, leading to the production of a neurotoxic protein, known as mutant huntingtin, with an abnormally long polyglutamine stretch. Even though the genetic background of HD is known, the cellular pathways affected in the disease are complex and not completely understood. Increasing evidence indicates that endoplasmic reticulum (ER) stress – a condition of disturbances in normal ER activity, leading to accumulation and aggregation of misfolded proteins in the ER lumen – is a central factor in the pathogenesis of HD and other neurodegenerative diseases. In the literature review of this thesis, known pathogenic cellular mechanisms of HD and how these cellular mechanisms are connected to ER stress, are discussed. Unpublished data from previous studies in our laboratory have indicated that the ER luminal protein canopy homolog 2 (CNPY2) could play a role in the regulation of neuronal survival, including the viability of mutant huntingtin expressing neurons. The aim of the experimental part of this study was to gain insight into a possible function of CNPY2 in HD, by examining the levels of the protein in neuronal models of HD under various conditions, such as ER stress, and by searching for potential interacting partners of CNPY2 amongst known ER stress regulators. The obtained results show that the levels of CNPY2 are increased in striatal neurons expressing mutant huntingtin, and that the secretion of CNPY2 is increased by these neurons, compared to control neurons expressing normal huntingtin. Further, we show that CNPY2 interacts with the major ER stress regulator binding immunoglobulin protein (BiP) in human neuroblastoma cells treated with the ER stress inducer tunicamycin, and that the intracellular levels of CNPY2 are altered by tunicamycin treatment. Together, these findings indicate that CNPY2 could be involved in the pathogenesis of HD. However, further research on the functions of CNPY2 and its role in ER stress regulation is required to understand the nature of this involvement.
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Comparison of The U.S. and Finnish pharmaceutical markets and their impact on health care spending (2020)Theoretical framework: The consolidated pharmaceutical market is becoming increasingly global and the same international pharmaceutical companies operate around the world in different countries, responsible for drug development and production. The high costs of developing novel medicines and the motive for higher profits has led to elevating price level of pharmaceuticals and health care services. Finland and the U.S. offer two extremes at the pharmaceutical market. The pharmaceutical market field in Finland is very structural and rigid, and medicine prices are regulated by law. In the U.S. the prices are based on the laws of supply and demand and the prices differ by different states, retailers and insurance policies. A small-scale longitudal price comparison is also reviewed to showcase the effect of continuously rising medicine prices. Study objective: The idea of this study is to describe and compare pricing mechanisms of pharmaceuticals and price differences between two very different market structures and review how these might affect the cost-effectiveness of national health care spending. These divergences are also mirrored to survey recent global pharmaceutical market problems such as drug shortages, possibly due to less appealing markets of higher price regulation policies. Materials and methods: Price data were collected from national, official, open-source databases. National health care expenditure and comparison to GDP was collected from publications by the OECD. All monetary values have been presented in both currencies (EUR and USD) to present more comparable values. Results: When compared to other OECD-countries the U.S. spent distinctly the largest amount of funds on health care per capita. Finland’s national health care costs were thousand times minor in total spending and less than a half per capita when compared to those of the U.S. With lower expenditure Finland manages to offer access to public, government-funded health insurance program. Meanwhile the prices of prescription medicines in Finland have decreased significantly, the prices for have continuously elevated in the U.S. Conclusions: The outcome of this study is that free markets and a complex supply chain, compared to more regulated markets with more transparency, have higher overall price level in pharmaceuticals and health care services. Free markets and sufficient intellectual property rights are more enticing to pharmaceutical companies. They promote new innovations and developing of much-needed novel therapies to modern health problems, such as AIDS and the global threat of worsening situation of antibiotic resistance. More regulated markets may create problems such as drug shortages and are often considered complex and less appealing market systems due to high level of administrative work but conserve the cost-effectiveness of the use of public funds.
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(2020)Parenteral products are sterile products that are administered as injection, infusion or implantation. Administration of the contaminated parenteral product can cause severe consequences such as sepsis meningitis and even death. Most of the parenteral products used at the hospitals needs to be compounded (e.g. dissolved, diluted) before administration. Whenever possible, compounding should be done in biological safety cabinet using aseptic techniques. According to previous studies errors in aseptic techniques are quite common. Aim of this study was to compare three different environments as compounding area and their effect to the sterility of the compounded parenteral product. Based on the results of this study, changes to the protocols of the hospital could be made. Altogether 220 samples were compounded at two pediatric wards at HUS Helsinki University Hospital. Six volunteers (one pharmacist and five nurses) participated from both wards and each compounded 18 samples in three different environments (patient room, medicine room, biological safety cabinet). The samples were tested for the sterility by membrane filtration within 4 hours or after 24 hours of storage in the refrigerator. The investigator used an observation form to observe the compounding procedures. Environmental monitoring (settle plates) and monitoring of personnel (glove samples) were conducted. Almost all compounded samples (99%, n=213/215) were sterile. There were no significant differences in the contamination rate of the compounded samples between different environments. Five of the collected samples were excluded, because they were contaminated during the sterility test. According to observations, aseptic techniques were well followed. However, disinfection of the septum of the medicine bottle, hand hygiene and cleaning of the compounding area were observed to be deficiently completed. Even though there were lot of variation in the environmental and personnel monitoring the results were quite good. Results from the environmental monitoring were compared to the recommended limits of EU GMP for clean areas. One compounded sample was contaminated with Diezia maris and Corynebacterium mycetoides but the contaminants from the other contaminated sample could not be identified. Aseptic techniques were mainly well followed, however compounding should be done in the biological safety cabinet, since the environmental monitoring results show that the biological safety cabinet was only environment which was within the recommendation limits of the EU GMP for the compounding area of parenteral products. Protocols of the hospital could be changed, since there was no correlation between higher contamination rate of settle plates or compounded samples and not wearing mask and hair cover while compounding in the biological safety cabinet.
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(2023)Orphan medicinal products (OMPs) are pharmaceuticals, that are utilized in the treatment of rare diseases. Rare diseases are diseases with a prevalence of at most five individuals out of 10 000. Clinical trials with statistically robust clinical data are challenging to conduct with rare diseases, since patient populations are small and the amount of trial subjects enrolling into these trials are usually scarce. Rare diseases also represent a variety of different diseases with divergent properties (5000-8000 identified). This presents challenges in health technology assessment (HTA) when reimbursements for these treatments are assessed and decided, especially when these treatments are usually considerably expensive and burdening to national health care systems. The main objectives for this study and master´s thesis was to research via interviews with experienced professionals from pharmaceutical industry and officials as how to define, monitor and assess the clinical effectiveness of OMP treatments, how to enhance their market access, and how to develop the current conditional reimbursement system in Finland. The interviewees (n=12) all represented from their respective backgrounds and introduced opinions from their own occupational positions and frameworks based on their professional experience. The study was executed as a qualitative study utilizing semi-structured interviews with predetermined questions and themes between 6th of April 2023 and 8th of June 2023. The interviewees were initially contacted via email and phone by one of this thesis supervisors from a professional pool of individuals identified using purposive sampling. The interview transcriptions were examined and analyzed using content analysis, and they were coded and grouped into themes. When inquired, the most common opinions regarding how to define and monitor clinical effectiveness of OMP treatments, the most common answers emphasized individual clinical assessment, real world data (RWD) collection, consideration of symptom control and overall quality of life, economic effectiveness, and clinical expert assessment. Market access of OMPs could be enhanced with more flexible and bold applications for negotiations and agreements, and a need for uniform, predictable MEA procedures, parameters for treatment discontinuation, outcomes-based models, and earlier proactive start for initial negotiations on behalf of the payer (society). The existing conditional reimbursement system might be developed with outcomes-based models, increased dialogue and trust between companies and officials within the realm of negotiations, uniform and predictable MEA procedures, and already established MEA negotiation frameworks to fast tract OMP market access. Development of the existing conditional reimbursement scheme, as well as objective to enhance market access environment in Finland could be accomplished by novel, flexible, patient specific, holistic and bold systems with an emphasis on systematic collection of RWD. Uniform and predictable MEA procedures with predetermined negotiation frameworks could bring value through faster market access and valuable predictability for pharmaceutical companies in their operations. Rapid market access of OMPs could be beneficial via clinical effectiveness of the treatments, as well as through collecting valuable clinical data from the medicinal products.
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(2024)Tutkimuksen tausta: Avohuollon apteekit tekivät monia toimenpiteitä varmistaakseen apteekkipalveluiden jatkuvuuden ja lääkkeiden saatavuuden COVID-19-pandemian aikana. Kriisijohtamisen prosessiteoria antaa rakenteellisen viitekehyksen kriisien ymmärtämiseen ja hallintaan. Tavoite: Tutkimuksen tavoitteena oli tutkia suomalaisten avohuollon apteekkien kriisijohtamisprosessia COVID-19-pandemian aikana käyttäen kriisijohtamisen teoriaa teoreettisena viitekehyksenä. Menetelmät: Poikkileikkauskyselytutkimus kehitettiin kriisijohtamisen prosessiteorian pohjalta ja lähetettiin suomalaisille avohuollon apteekkareille sekä yliopistoapteekkien johtaville proviisoreille toisen pandemia-aallon aikana loka–marraskuussa 2020. Logistisen regressioanalyysin avulla tutkittiin, oliko johdon riskinäkemyksellä yhteyttä kriisivalmiuteen, ja miten kriisisuunnitelmat, tiimit ja yhteistyö ulkoisten sidosryhmien kanssa olivat yhteydessä pandemian vaikutuksiin avohuollon apteekeissa. Avoimista vastauksista saatu laadullinen aineisto ryhmiteltiin samankaltaisuuksien perusteella. Tulokset: Kyselyyn vastasi yhteensä 221 apteekkaria ja johtavaa proviisoria (vastausprosentti 36,7 %). Apteekeista 79,6 %:lla oli olemassa pandemiasuunnitelma ennen kriisiä. Pandemiakriisitiimi oli nimetty 35,3 %:lla apteekeista ja 33,5 % lisäsi tai paransi yhteistyötä muiden apteekkien tai lääkehuollon toimijoiden kanssa. Kollektiivinen päätöksenteko ja ulkoisen yhteistyön lisääminen tai parantaminen olivat yhteydessä vähäisempiin negatiivisiin vaikutuksiin johdon jaksamisessa. Lisäksi kollektiivinen päätöksenteko oli yhteydessä vähäisempiin negatiivisiin vaikutuksiin organisaation taloudessa. Kirjallisuudesta poiketen olemassa olevalla pandemiasuunnitelmalla oli enemmän negatiivisia vaikutuksia organisaatioiden resursseihin. Yhteyttä apteekkarin riskinäkemyksen ja apteekkien kriisivalmiuden välillä ei havaittu. Johtopäätökset: Apteekkien tekemät toiminnalliset muutokset ja infektion ehkäisy- ja torjuntakäytännöt mahdollistivat keskeisten palvelujen tarjoamisen pandemian asettamista haasteista huolimatta. Oppimista ja sopeutumista tapahtui reaaliajassa kriisin aikana. Kollektiivisen päätöksenteon kehittäminen ja yhteistyö kollegoiden ja muiden lääkehuollon toimijoiden kanssa voivat parantaa apteekkarien jaksamista ja apteekkien taloutta tulevissa kriiseissä. Jatkotutkimuksissa voitaisiin hyödyntää laadullisia tutkimusmenetelmiä ja tutkia tarkemmin kollektiivista päätöksentekoa sekä vaikuttavan kriisisuunnitelman sisältöä avohuollon apteekkien kontekstissa.
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(2020)Background: Alzheimer’s disease (AD) is a worldwide challenge for health care professionals and researchers. Every year, AD causes dementia for millions of patients. No preventive or curative medication is available despite continuous research. Amyloid-beta (Aβ) deposits in brain are one of the main pathological findings in AD. Accumulating Aβ peptides are thought to be the reason behind further disease progression. If the Aβ accumulation could be restricted or Aβ degradation increased their toxic effects would be prevented. Soluble oligomers and protofibrils are the most toxic species of Aβ. Most of the Aβ targeting drugs developed so far have not specifically targeted these toxic species. Neprilysin (NEP) is a major Aβ degrading enzyme that targets mostly the smallest species (monomers and dimers) of Aβ. Another common challenge for protein drugs has been passing the blood-brain barrier (BBB). Different strategies, such as utilising transferrin receptor (TfR) mediated transcytosis, have been studied for drug transport. For example, a rat anti-mouse TfR antibody, 8D3, or its fragments can be used for drug transportation. Objectives: To produce a recombinant protein, sNEP-scFv8D3, combining soluble NEP and single chain variable fragment of 8D3. Testing its ability to degrade different species and isoforms of Aβ in vitro and study in vivo brain uptake. Evaluate whether it is a promising model for future AD treatments. Methods: The recombinant protein was expressed in Expi293 cells and purified with affinity chromatography. The TfR binding was studied with TfR ELISA and enzymatic activity with MCA assay. Aβ ELISA was used for determining the Aβ degradation. Recombinant protein was compared to sNEP. In in vivo studies the brain uptake and blood half-life of radiolabeled sNEP-scFv8D3 of were studied on NLGF mice. Immunohistochemical analyses of brain cryo sections were done to evaluate the co-localisation of Aβ aggregates and sNEP-scFv8D3. Results and discussion: sNEP-scFv8D3 bound to TfR and showed similar enzymatic activity as sNEP. Both sNEP-scFv8D3 and sNEP were able to degrade monomeric Aβ-40 and Aβ-42 but no significant effect was seen on larger aggregates. In mice brain, sNEP-scFv8D3 was detected in same areas as Aβ aggregates. Compared to sNEP, our recombinant protein had better brain uptake. The blood half-life of sNEP-scFv8D3 was approximately 9.5 h and it was cleared fast from the brain. Already 6 h post injection, levels in the brain had dropped more than by half. Further studies are needed to determine whether sNEP-scFv8D3 is effectively transported across the BBB and if it can reduce brain Aβ levels in vivo. Conclusions: In the future, sNEP-scFv8D3 or its improved version could be used at the earliest stages of AD to prevent disease progression. Since sNEP-scFv8D3 degrades only small Aβ aggregates it could be combined with another drug targeting larger oligomers. Together they would decrease the total Aβ deposition in brain.
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(2020)Parkinson’s disease (PD) is a neurodegenerative disease in which dopaminergic neurons that form the nigrostriatal pathway gradually die. This causes the main motor symptoms of Parkinson’s disease: tremor, rigidity and bradykinesia. While PD affects 1-2% of total population, all currently used medicines are symptomatic, and there is no disease modifying therapy available at present. Although several different animal models for Parkinson’s disease exist, the lack of adequate animal models is often cited as a major obstacle for predicting the clinical success of potential drug candidates. Lewy bodies (LBs) are abnormal aggregates that develop and spread inside nerve cells of human PD patients, their main structural component being α-synuclein. Because α-synuclein is thought to play a major role in the pathology of PD, much research has been focused on it. Different α-synuclein-based animal models of PD exist today, of which the most recent are based on using direct injections of preformed α-synuclein fibrils (PFFs). These new α-synuclein based disease models have helped to understand the disease process in PD better, but cell death in these models takes longer to achieve and is often less pronounced compared to traditional neurotoxin based animal models of PD. The aim of this study was to participate in the development and characterization of a novel mouse model of PD. This new model combines PFF-injections with the commonly used neurotoxin 6-OHDA, which should result in more robust dopamine pathway degeneration than what is seen with the current PFF-based models. The main hypothesis of this study was that the combination of intrastriatal injections of PFFs and a low dose of 6-OHDA would cause gradual spreading of the α-synuclein aggregation pathology in the nigrostriatal dopamine pathway and progressive dopamine neuron loss leading to motor deficits. C57BL/6 mice were stereotactically injected unilaterally with both PFF and 6-OHDA, and their performance was assessed every other week with different behavioral tests until week 12. At the end, brains were collected and optical density of tyrosine hydroxylase (TH) and dopamine transporter (DAT) was measured from striatal sections, and TH and DAT positive cells in the substantia nigra were counted. The amount of Lewy bodies present in the brain slices was also counted from the cortex and substantia nigra areas of the brain. In the histological assays, statistically significant reductions of both TH and DAT were found in the brain sections of the PFF + 6-OHDA combination group and the amount of TH and DAT positive cells were lower in this group compared to the group receiving vehicle treatment only. However, the results of behavioral tests were non-significant, although a non-statistical positive trend in the amphethamine-induced rotations test was observed where mice receiving PFF + 6-OHDA rotated the most. Taken together, combination model that utilizes both PFF and 6-OHDA injections seems like a promising candidate in modelling PD in mice, but much more research and further development of the model is required before this combination model is ready and robust for use in drug development.
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(2021)Mild traumatic brain injury (TBI) is defined as an injury that disrupts the normal functioning of the brain and is the result of external force to the head. It is the most common type of traumatic head injury, and it is common especially in contact sports and within military personnel. Mild TBI typically causes no clear structural changes to the head, but it can induce persistent clinical symptoms, as well as microscopic pathological changes to the brain that may eventually lead to neurodegeneration and increase the risk for several diseases. Mild TBI is a risk factor for several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and chronic traumatic encephalopathy. The primary objective of this study was to develop a repetitive mild TBI mouse model for future research purposes in the field of head trauma and neurodegeneration. The injury was induced as a closed head injury with an electromagnetic impactor. Literature and pilot experiments were used to define the parameters of the impactor required to induce a brain injury of desired severity. The characterization criteria of the mild TBI model considered the criteria used to define human mild TBI, as well as long term effects often reported after repetitive mild TBI: neurodegeneration as tau protein related pathology, neuroinflammation, and memory deficits. The secondary objective of this study was to tentatively test a prolyl oligopeptidase (PREP) inhibitor on the behavioral and histological effects of mild TBI. The functioning of the mild TBI model was studied by histopathological and behavioral assessments. After baseline behavioral assessment and repetitive (1 injury every 24 hours altogether 5 times) mild TBI inductions, the mice were monitored for approximately 3 months, during which several rounds of behavioral tests were performed. Barnes maze and novel object recognition tests were used to assess memory functions, and locomotor activity test was used to assess general locomotor activity. After euthanasia, brain histopathology was performed to study the amount of tau protein and the level of neuroinflammation. Due to the low number of animals in the study, the results are directional and need to be confirmed in subsequent studies. The histopathology showed greater amount of neuroinflammation and tau protein in the brains of injured mice, but statistical evaluations could not be made. Memory functions were slightly worse in the injured mice compared to controls, but significance of the results is unclear. Locomotor activity was not influenced by the mild TBIs. PREP inhibition treatment increased the locomotor activity of the mice, but the significance is unclear. The mild TBI model seems promising and the characterization criteria were partially met. The results of the study need to be verified in subsequent studies with a greater amount of animals. The model developed here can be used to study the involvement of head trauma in neurodegeneration, as well as treatment alternatives to changes caused by mild TBIs. As there currently are no curative treatments to neurodegenerative diseases, research regarding neurodegeneration and its risk factors is highly important.
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(2021)Cytochrome P450 (CYP) enzyme inhibition is one of the most common reasons for adverse drug-drug interactions. An especially harmful form of inhibition is time-dependent inhibition (TDI) in which the inhibition potency increases over time and persists even after discontinuation of the drug. Both direct and time-dependent inhibition can be efficiently screened with the so-called cocktail method containing several CYP-selective probe substrates in a single reaction mixture. This method is practical especially in ADME studies of drug development, as it offers lower costs, consumption of fewer reagents and faster implementation in comparison to conventional methods. In addition, the cocktail method can be used to establish new diagnostic CYP inhibitors in vitro. The aim of this Master’s thesis was to participate in the development and optimization of a new cocktail assay method. The method was developed for screening of major drug-metabolizing CYP enzymes in vitro both in a direct and time-dependent manner using pooled human liver microsomes. Based on preliminary testing, included probe substrates were divided into two cocktails to avoid significant inter-substrate interactions: cocktail I containing tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4, and cocktail II containing coumarin/CYP2A6, (S)-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2. First, cocktail incubation conditions were optimized, followed by the determination of probe reaction kinetics, kinetic parameters (Km, Vmax) and inter-substrate interactions with single- or dual-substrate incubations. Finally, suitable probe substrate concentrations and the composition of cocktails was evaluated based on the obtained results. As a result of assay optimization, optimal incubation conditions for yet unoptimized cocktail II were established. In optimized incubation conditions, all probe reactions exhibited saturable Michaelis-Menten kinetics except for tacrine 1-hydroxylation (CYP1A2), which exhibited biphasic kinetics instead (Km1: 7.36, Km2: 517). The selected probe substrate concentrations were all below or near their respective Km values except for (S)-mephenytoin 4’-hydroxylation (40 µM vs. Km of 12.5 µM); however, its concentration could not be reduced in order to maintain sufficient metabolite formation for UHPLC-MS/MS-analysis. Dual-substrate incubation assays demonstrated a need for the reduction of bupropion concentration below 100 µM due to its inhibitory effects on CYP2C8 and CYP3A4. In addition, chlorzoxazone/CYP2E1 and testosterone/CYP3A4 were tested as complementary probe substrates for the cocktails; however, they proved to be unsuitable for both cocktails due to significant interactions (>40% inhibition). Prior to the deployment of the method, some adjustments of probe substrate concentrations are still required in addition to consideration of the suitability of less commonly used CYP3A4 and CYP2E1 probe reactions to improve cocktail coverage. Lastly, validation of the method with known time-dependent model inhibitors should also be conducted. Besides to improvement of the cocktails, new information was generated on inter-cocktail probe-probe interactions and enzyme kinetics of probe reactions, especially for the less-studied astemizole O-demethylation (CYP2J2) and tacrine 1-hydroxylation (CYP1A2). Generated information can be used, for example, in the development of new cocktails.
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(2021)Background and objectives: Cancer is one of the leading causes of death worldwide, and resistance to current treatments demands the continuous development of novel cancer therapies. Cancer immunotherapy aims to induce anticancer immune responses that selectively target cancer cells. Viruses can also be harnessed to elicit tumor-specific immune responses and to improve the response rates of other concomitant cancer therapies. The purpose of this study was to develop a novel viral vector-based cancer vaccine for intratumoral immunotherapy. By using the previously developed PeptiENV cancer vaccine platform, the vector viruses were coated with cell-penetrating peptide (CPP) sequence-containing tumor peptides in an attempt to further drive the immune responses elicited by the vector against cancer cells. The efficacy of the PeptiENV complex as a cancer vaccine was assessed by following its effects on tumor growth and the development of local and systemic antitumor immune responses. Methods: The PeptiENV complex formation was assessed by a surface plasmon resonance (SPR) analysis. Dendritic cell (DC) activation and antigen cross-presentation were studied using the murine JAWS II dendritic cell line. The development of cellular immune responses against tumor antigens was first studied by immunizing mice with the PeptiENV complex. The antitumor efficacy and immunity of intratumoral PeptiENV administration were then studied using the murine melanoma models B16.OVA and B16.F10.9/K1. In addition to intratumoral PeptiENV treatment, some of the B16.F10.9/K1-implanted mice were also treated with an anti-PD-1 immune checkpoint inhibitor (ICI) to study the PeptiENV complex as a biological adjuvant for ICIs. Results: The SPR analysis confirmed that CPP-containing peptides can be stably anchored onto the viral envelope of the viral vector. The in vitro results showed that the PeptiENV complex does not hamper the presentation of antigens at the surface of DCs. Additionally, the viral vector was found to activate DCs seen as a change in the cells’ morphology and surface protein expression. Immunizing mice with the PeptiENV complex induced a robust antigen-specific cytotoxic T cell response. Upon intratumoral administration in vivo, the PeptiENV cancer vaccine was not capable of inducing tumor growth control against B16.OVA melanoma, although it did still elicit robust systemic and local antitumor T cell responses. In the treatment of B16.F10.9/K1 melanoma, however, the PeptiENV complex induced efficient tumor growth control, which resulted in a significant survival benefit. Additionally, co-administration of anti-PD-1 resulted in an additive therapeutic effect. Discussion and conclusions: The present study describes a novel, highly immunogenic viral vector-based cancer vaccine that has the potential to be used as an adjuvant treatment for ICI therapy. Subsequent studies could be conducted to gain a deeper understanding of the immunological mechanisms underlying the antitumor efficacy of the cancer vaccine complex. Moreover, this novel PeptiENV complex could also be further developed as an infectious disease vaccine platform against emerging pandemics. However, the effects of pre-existing antiviral immunity on the efficacy of the cancer vaccine should be explored in future studies.
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(2024)The potential of extracellular vesicles (EVs) as diagnostic markers and drug delivery vehicles has been studied increasingly in recent years. One of the challenges in this field has been the isolation of EVs from complex biological fluids such as blood. The methods widely used for the isolation process include for example size exclusion chromatography (SEC) and ultracentrifugation (UC). As these methods use size and density of the particle, the have not been efficient enough in isolating EVs from certain particles such as lipoproteins. Due to the challenges related to these methods, other isolation methods have been sought to improve the efficiency of EV isolation. One of these methods is ion-exchange chromatography (IEC). From the two forms of IEC, anion-exchange chromatography has been studied more in EV isolation due to the negative net charge on EV particles. However, in this study the functionality and efficiency of cation-exchange chromatography (CEC) in EV isolation was studied as very little research has been done on this method. In this study, two CEC-resins were studied to define their applicability in EV isolation. A standard strong cation-exchange chromatographic resin SP Sepharose Fast Flow was compared to a strong tentacle-type resin. In addition to this, we studied the possibility to use a magnesium gradient to separate different forms of lipoproteins from EVs through dextran-sulfite precipitation. Tentacle-type CEC-resin was found to be more efficient in capturing EVs compared to the standard-type resin without magnesium. These EVs could then be eluted from the column with sodium chloride. The use of magnesium gradient allowed the separation of apolipoproteins in the samples. Higher concentrations of magnesium also reduced the number of lipoproteins in the samples altogether but resulted in the loss of EVs as well. These results were promising and showed that cation-exchange chromatography can be used in EV isolation. Tentacle-type resin seemed to be most efficient in removing impurities and capturing EVs. While more research is needed before these findings can be applied to clinical use, these results prove that cation-exchange chromatography can be used in EV isolation as a new, efficient and up scalable method.
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(2021)Drug metabolism is a series of enzyme catalysed processes that modify foreign compounds into a form that is more easily excreted from the body. Compounds can affect the activity of metabolizing enzymes and this may lead to toxic concentrations of a drug that is metabolized via the enzyme. With prodrugs, on the other hand, the drug might not achieve its biologically active form and therefore the treatment will not be effective. Recognizing and preventing metabolic interactions is important already in the early stages of drug discovery and development. Cytochrome P450 (CYP) enzyme inhibition is one of the major reasons for adverse drug-drug interactions (DDIs). The inhibition can be time-dependent (TDI), which means that the potency of inhibition increases over time. TDI may be reversible or irreversible, latter being more severe as new enzymes need to be produced in the body to restore the enzymatic activity. IC50 shift assay is a method that gives information of new compounds potential to cause TDI. IC50 shift assay does not show whether the TDI is reversible or irreversible, however further studies, e.g. dialysis assay, can be conducted to find it out. If the study compound is irreversibly bound to the enzyme, the enzyme activity should not recover in the dialysis. The aim of this master’s thesis was to develop a dialysis method that could determine the reversibility of the TDI observed in the IC50 shift assay. A dialysis method conducted with microsomes is described in earlier literature. Known inhibitors (both time-dependent and direct) for four CYP isoforms were studied in this work: CYP1A2 (furafylline and fluvoxamine), CYP2C9 (tienilic acid and sulphaphenazole), CYP2D6 (paroxetine and quinidine) and CYP3A4 (verapamil, azamulin and ketoconazole). IC50 shift assays were conducted to each inhibitor before the dialysis experiment. The studied compounds behaved in the dialysis assay mostly as assumed based on the literature. The workflow from IC50 shift assay to dialysis assay worked successfully and the IC50 shift data could be utilized when choosing the test concentrations for dialysis assay. Both the IC50 shift assay and dialysis assay were reproducible and the deviations between replicates and separate studies were relatively low. The method still requires some optimizing, but so far, the results are promising. In the future the dialysis method may be part of in vitro CYP inhibition studies at Orion Pharma.
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(2021)Neurodegenerative diseases and neuronal injury after trauma are common causes of neuronal loss. Adult brain has only a limited regenerative capability to replace the lost neurons caused by several distinct brain diseases. Direct reprogramming of brain resident cells into neurons could provide a promising strategy for efficiently replacing non-functional neurons. To date, the focus has been put largely on astrocyte-to-neuron reprogramming despite the relatively low yield of newly generated neurons reported in vivo. According to our hypothesis oligodendrocytes possess a more diverge transcriptomic profile when compared to neurons and astrocytes thus allowing better cell-specific targeting of reprogramming. Here, we establish the molecular tools for direct neuronal reprogramming of human oligodendrocytes to neurons. We investigate whether the expression of a known neural fate specification factor under selected oligodendrocyte-specific promoters is sufficient to induce oligodendrocyte-to-neuron transformation. Furthermore, we test the established tools in vitro using an immortalized human oligodendrocyte cell line. Our preliminary data shows that the human ERBB3 promoter and a single transcription factor transfected cells express doublecortin (DCX), an early marker of neuronal identity. Only recently, the direct in vitro reprogramming of human oligodendrocyte precursor cells into functional neurons has been reported. The direct reprogramming of oligodendrocytes into neurons provides an exciting alternative of neuronal replacement for astrocyte-to-neuron reprogramming. Overall, the field of direct reprogramming offers interesting possibilities for regenerative medicine providing a method for the production of newly generated disease and patient-specific cells.
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(2024)New drugs against malaria are required, as millions of people are still affected yearly by this deadly disease. The development of drug resistance to current antimalarials is an ongoing process. Membrane-bound pyrophosphatases (mPPases) are potential new drug targets against malaria and other protozoan diseases. mPPases play a crucial role in the survival of the malaria parasite, they couple the energy released from the hydrolysis of pyrophosphate into the transport of protons or ions against an electrochemical gradient. The aim of this study was to identify potential mPPase inhibitors through a docking-based virtual screen of the Tres Cantos Antimalarial Compound Set, which consists of over 13500 malaria-active compounds. The virtual screen against a Thermotoga maritima mPPase protein structure identified a 2,4-diamino-1,6-dihydrotriazine among the top-ranking scaffolds. Four compounds found among the docking results containing this scaffold were synthesised: three with a halophenyl substituent, and one with a hydroxyl substituent. The compounds in their hydrochloride salt forms were synthesised using a three-component method for the synthesis of 2,4-diamino-1,6-dihydrotriazines. The compounds were also freed from the hydrochloride salts into their corresponding molecular forms. The structural characterisation of the compounds, especially the molecular forms, presented challenges. The docking results were also searched to identify compounds containing previously identified mPPase-active substructures. From the docking results, several other interesting compounds were identified in addition to the synthesised compounds. The knowledge and results obtained from this study can be used as openings for potential future docking and synthesis projects in the development of mPPase inhibitors. The activity of the compounds synthesised in the project remains to be evaluated in subsequent investigations.
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(2024)Congenital heart diseases develop during heart development and encompass structural abnormalities in the heart present at birth, with hypoplastic left heart syndrome (HLHS) representing a rare but life-threatening subtype. HLHS is characterised by the underdevelopment of left-sided heart structures, resulting in a major blood flow obstruction of the heart, impairing systemic circulation. Current knowledge of HLHS aetiology is scarce, which makes the development of effective treatments challenging. Therefore, identifying the disease mechanisms causing HLHS is essential. Notably, HLHS is linked with mutations in the NKX2-5 gene, which encodes for a cardiac transcription factor and has a pivotal role in heart development together with the transcription factor GATA4. This makes these genes intriguing research targets in HLHS. This study aims to enlighten how HLHS patient-derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) differ from those derived from healthy donors in terms of stress response by subjecting hiPSC-CMs to pro-hypertrophic stimuli, namely endothelin-1 (ET-1) and cyclic mechanical stretching. Additionally, the effects of GATA4-targeted compounds on these hypertrophy models were also studied, which included two inhibitors (3i-1262 and 3i-1000) and one activator (3i-0777) of GATA4-NKX2-5 interaction. Differentiation of CMs was performed using a small-molecule induction protocol based on sequential Wnt pathway activation and inhibition. The effects of ET-1 and cyclic mechanical stretching were analysed by High-content analysis for pro-B-type natriuretic peptide (proBNP) expression, and quantitative PCR for hypertrophic gene expression, respectively. Both ET-1 and cyclic mechanical stretching effectively induced hypertrophy in their respective models. This was observed in all cell lines as a higher hypertrophic response of proBNP in ET-1 exposed hiPSC-CMs and upregulation of hypertrophic genes NPPA and NPPB in stretched hiPSC-CMs. GATA4-targeted compounds did not show statistically significant effects on ET-1-induced hypertrophy or stretching-induced hypertrophic gene expression in any cell line, but various trends could be distinguished. As expected, both inhibitor compounds, 3i-1262 and 3i-1000, showed a tendency for antihypertrophic effects since they decreased the percentage of proBNP+ cells in all cell lines. Unexpectedly, the activator compound 3i-0777 also decreased the percentage of proBNP+ cells. We also observed that HLHS-disease cell line HEL 149 seemed to differ from the three other cell lines showing a phenotype that exhibits similar gene expression patterns as seen in heart failure patients. This was mainly observed as a statistically significantly lower basal MYH6 gene expression. However, the limited experimental setup of this study requires further experiments to detect significant differences and draw definitive conclusions regarding the effects of GATA4-targeted compounds on hypertrophic stimuli.
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(2021)Dilated cardiomyopathy is a non-ischemic cardiac disorder predisposing to heart failure, and the characteristics of dilated cardiomyopathy emerge under normal loading conditions. Dilated cardiomyopathy can be consequence of various conditions e.g. genetic mutations, virus infection or toxin exposures. One of the significant causes of familial dilated cardiomyopathy in Finland is mutation S143P in LMNA-gene, coding for A type lamins. Current drug therapy for dilated cardiomyopathy aims to alleviation of symptoms, prevention of complications and progression of the disease, however, efficacy of current therapy is insufficient, and novel therapy strategies are urgently required. Transcription factors are fundamental regulators of gene expression, and GATA4 is a crucial transcription factor both in embryonic and in adult heart and thus an intriguing target for therapeutic manipulation. Compounds targeting GATA4 have shown anti-hypertrophic and cardioprotective effects. Here, effects of two different hypertrophic stimuli, endothelin-1 and mechanical stretch, on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were examined with high-content analysis and quantitative reverse transcription PCR (qRT-PCR), respectively. One hiPSC-CM line was used as a healthy control, whereas the other carried the S143P mutation in LMNA-gene (DCM-CMs). Additionally, effects of GATA4-targeting compound C-2021 on cardiomyocytes were investigated. In summary, according to proBNP staining, DCM-CMs are more hypertrophied at baseline. DCM-CMs seemed to be less susceptible to mechanical stretch-induced enhancement in BNP gene expression. In addition, compound C 2021 may have anti-hypertrophic properties suggesting it to be a potential drug candidate in cardiac diseases. Finally, lamin A seemed to mislocalize to nucleoplasm instead of nuclear lamina in DCM-CMs.
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(2024)Parkinson’s disease (PD) is a progressive neurodegenerative disorder in which dopaminergic neurons in the substantia nigra (SN) degenerate and die. This causes multiple motor symptoms such as rigidity, bradykinesia and tremor and non-motor symptoms such as depression, hallucinations, and cognitive impairment. At the time of the diagnosis, approximately 60% of the dopaminergic cells can already be lost, which underlines the importance of neurorestorative treatments for PD. Currently used treatments are only symptomatic and mostly based on levodopa, which can lose its effectiveness as the disease progresses and additionally cause significant side effects such as dyskinesia. Neurotrophic factors (NTF) such as glial cell-line derived neurotrophic factor (GDNF) and neurturin (NRTN) have been studied in clinical trials with PD patients but have shown only modest effects on motor function. Additionally, they have been administered with invasive techniques such as intraputamenal or intracerebroventricular injections which includes many risks. Mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to unconventional NTF’s with unique molecular structure and mode of action. MANF has shown both neuroprotective and neurorestorative properties for nigrostriatal dopamine system in in vivo study in rat model of PD. To enable systemic administration of MANF, the molecule has been modified by retaining only the C-terminal domain to form C-terminal MANF fragment (C-MANF). C-MANF has shown neurorestorative effects when administered intrastriatally in 6-OHDA lesioned rats and when injected subcutaneously (s.c.) in an ALS animal model. The aim of this study was to assess the effect of daily and weekly s.c administered C-MANF in 6-OHDA mouse model of PD. Cylinder and amphetamine-induced rotation tests were performed as behavioural tests and thereafter morphological studies were done by analyzing tyrosine hydroxylase (TH)+ cells in the substantia nigra pars compacta (SNpc) and optical density from TH+ axons in the striatum. Interestingly, weekly C-MANF treatment decreased the number of TH+ cells in SNpc and the density of TH+ fibers in the striatum compared to PBS. However, it decreased ipsilateral rotations and showed some positive effects in the cylinder test. On the other hand, daily C-MANF treatment increased the number of TH+ cells in SNpc and the density of TH+ fibers in the striatum but had a modest effect on ipsilateral rotations and the cylinder test compared to PBS. Although no statistically significant effects were observed in behavioral and morphological studies, s.c administered C-MANF presents a promising treatment option for PD. Particularly, daily administration of C-MANF showed neurorestorative effects in morphological studies; however, further research is required for validation. Additionally, the investigation of higher doses of C-MANF should be considered.
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(2020)Lääkevaihto ja sitä täydentävä viitehintajärjestelmä ovat laskeneet lääkekustannuksia Suomessa. Epilepsialääkkeet eivät ole aiemmin kuuluneet lääkevaihdon piiriin, sillä epilepsian hoidossa eri valmisteet eivät välttämättä ole terapeuttisesti tarpeeksi samanarvoisia, ja pienikin muutos hoitotasapainossa voi altistaa epilepsiakohtauksille. Nykyisin epilepsialääkkeitä käytetään kuitenkin usein muihinkin käyttöaiheisiin, kuten psykiatrisiin sairauksiin ja kivun hoitoon. Vuonna 2017 lääkekorvausjärjestelmään tehtiin säästötoimenpiteitä, joiden yhteydessä epilepsialääkkeet sisällytettiin lääkevaihdon piiriin muissa käyttöaiheissa kuin epilepsian hoidossa. Lisäksi otettiin käyttöön poikkeava viitehintaryhmä, joka koski epilepsialääkkeistä pregabaliinia neuropaattisen kivun käyttöaiheessa. Tutkimuksen tavoitteena oli tarkastella epilepsialääkkeiden (pregabaliinin, gabapentiinin, topiramaatin, lamotrigiinin ja valproiinihapon) vaihtamista sekä hintojen kehitystä lääkevaihtoon ja viitehintajärjestelmään sisällyttämisen jälkeen vuoden 2017 alusta vuoden 2019 puoliväliin. Lisäksi tarkasteltiin näiden lääkeaineiden kustannusten, korvausmenojen sekä käyttäjä- ja reseptimäärien kehitystä. Aineistona käytettiin Kansaneläkelaitoksen reseptirekisteriin pohjautuvia tilastoja epilepsialääkkeiden lääkeostoista sekä lääkkeiden hintalautakunnan päätöksiä epilepsialääkkeiden viitehintaryhmistä ja viitehinnoista. Epilepsialääkkeiden vaihtaminen yleistyi tarkastelujakson aikana kaikilla lääkevaihdon piirissä olleilla viidellä lääkeaineella, ja vaihtokieltojen osuus resepteistä laski useimmilla lääkeaineista. Viitehinnat laskivat useimmissa tarkastelluista viitehintaryhmistä, mutta lähes yhtä usein viitehinta ei muuttunut. Viitehinnat laskivat enemmän viitehintaryhmissä, joissa oli useampia vaihtokelpoisia valmisteita. Lääkevaihdon ensimmäisenä vuonna 2017 lääkevaihtoon kuuluvien epilepsialääkkeiden kustannukset ja korvausmenot pääosin laskivat, vaikka lääkkeiden käyttö ei vähentynyt. Lääkevaihdon toisena vuonna kustannukset eivät juuri laskeneet. Pregabaliinin poikkeavan viitehintaryhmän vuoksi vaihtamatta jääneet reseptit aiheuttivat merkittävän osan lääkevaihtoon kuuluvien epilepsialääkkeiden kustannuksista. Pregabaliinille jäi siten todennäköisesti yhä säästöpotentiaalia poikkeavan viitehintaryhmän voimassaolon päätyttyä vuoden 2019 heinäkuussa, mitä on syytä tarkastella jatkotutkimuksissa.
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