Browsing by Subject "pharmacy"

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  • Hyytinen, Heidi; Siven, Mia; Salminen, Outi; Katajavuori, Nina (2021)
    Students in higher education have been shown to have difficulties in developing their critical thinking skills, such as analysis and problem solving, reasoning and argumentation. Open-ended tasks offer opportunities for students to develop their own interpretations of various sources, to critically analyse domain-specific knowledge and utilize that knowledge in their argumentation. This study focuses on the ability of new Master's students (n=37) to utilize pharmaceutical knowledge from different sources in producing written arguments and counter-arguments in the context of open-ended assignment task. The data were analysed by qualitative content analysis. The results showed that there was substantial variation in how students analysed and processed pharmaceutical knowledge as well as how they utilized that knowledge in their argumentation. While some students were able to provide comprehensive analysis of the different sources, others superficially analysed and processed the sources and struggled to generate convincing arguments. Students' written responses were typically one-sided: only a few students provided counter-arguments associated with the pharmaceutical problem-solving situation presented in the task. Understanding the nature of the challenges in argumentation and knowledge processing encountered by pharmacy students can help pharmacy educators to modify their pedagogical practices to better support students' learning. Practitioner Notes 1. University students even in Master program level may have challenges related to argumentation and processing knowledge 2. The challenges in argumentation and processing knowledge should be taken into account and should be enhanced and practiced from the beginning of the studies. 3. Critical thinking and argumentation should be integrated into the intended learning outcomes, learning and teaching activities, the contents of the courses, and assessment.
  • Subrizi, Astrid (Helsingin yliopisto, 2014)
    Biologics are increasingly used in the treatment of ocular diseases such as age-related macular degeneration (AMD) that cannot be controlled with conventional small molecule drugs. AMD is a multifactorial eye disease that carries significant risk of morbidity and vision loss. In Finland and other western countries, AMD affects one in three people older than 75 years, and until the early 2000s no effective treatment was available for these patients. The marketing approval of anti-VEGF antibodies was a major breakthrough in the management of AMD; indeed these biologics effectively halt choroidal neovascularization and therefore prevent further vision loss in roughly half of the patients with wet AMD. Antibody therapy has been the most successful approach so far, however, other biological therapies such as gene therapy, cell therapy and other therapeutic proteins, may prove beneficial in the treatment of AMD and other vision threatening disorders. This thesis deals with the delivery of biologics, including DNA, cells, proteins and peptides, to the retinal pigment epithelium (RPE), which plays a central role in the development of AMD. Briefly, the main topics and results of this work are presented. New non-viral gene delivery candidates are usually screened for transfection efficiency and toxicity by reading out transgene expression levels relative to a reference formulation after in vitro transfection. The screening protocols, however, can be very different among laboratories, so that comparison of results is often difficult, if not impossible. Our aim was to develop a standardized protocol optimized for the transfection of retinal pigment epithelial cells in vitro. The developed screening protocol provides a relatively simple and reproducible procedure for the pre-selection of potential candidate reagents as non-viral gene delivery systems targeted to the retinal pigment epithelium. The ocular delivery of biologics remains a challenging task due to the barriers of the eye. Short cationic peptides, also known as cell-penetrating peptides (CPPs), have been successfully used as tools to introduce various biologics into cells due to their ability to translocate across the plasma membrane and deliver their cargoes intracellularly. In our work, we have explored the functionality of Tat peptide, one of the most widely studied CPPs. Our results indicate that it is not the sequence of Tat per se that dictates cell uptake, but the cationic charge of the peptide. Moreover no direct penetration was observed; instead all the peptides were endocytosed and, as it is often the case in non-viral gene delivery, ended their journey inside lysosomes. For this reason, we think that the use of Tat peptide for the delivery of biologics to the cytoplasm or nucleus of cells will probably not be very successful. Ocular stem cell therapy holds promise for the reconstruction of the degenerated RPE monolayer in AMD patients; in addition, engineered human RPE constructs may also provide a unique platform for drug discovery and toxicology. We have grown a functional RPE tissue in vitro by using human embryonic stem cells as cell source and the synthetic polymer polyimide as supporting scaffold for the growth and maturation of the cells. The epithelia acquired RPE-like properties, including characteristic RPE phenotype, expression of RPE markers, barrier and phagocytic function. The degeneration of RPE cells in dry AMD is caused by the aggregation of proteins inside RPE cells, and is currently untreatable. We have investigated the cytoprotective properties of heat shock protein 70 kDa (Hsp70) against oxidative damage and the feasibility of rhHsp70 protein therapy as a potential therapeutic approach for dry AMD. This work provides a novel therapeutic option for the treatment of RPE degeneration in AMD.
  • Zanjanizadeh Ezazi, Nazanin (Helsingin yliopisto, 2021)
    Biomaterials science denotes a multidisciplinary science combining materials engineering, biomedical engineering, and biology. Tissue engineering is defined as an engineering system field using biomaterials to regenerate, restore tissue, or improve its function. This thesis focuses on the fabrication of scaffolds and patches for bone and heart regeneration using a conductive polypyrrole (PPy) polymer. PPy is widely used in different tissue engineering applications due to its biocompatibility, high electrical properties and conductivity, ease to synthesize, and environmental stability, such as in water and air. First, the role of this polymer in attracting protein and osteoblast cells was investigated. The bone scaffold’s mechanical properties using PPy were analyzed. The results showed no difference between conductive and non-conductive bone scaffolds, presenting almost the same young modulus between the two systems. In addition, more proteins were adsorbed on the surface of the conductive polymers. The same results were obtained in conductive cardiac patches, which showed higher blood coverage on the conductive 2D patch using PPy compared to the non-conductive one. In addition, cellulose was used to 3D print and fabricate a porous conductive cardiac patch. The in vitro investigations showed that cell attachment and proliferation on the conductive bone scaffold and cardiac patches were higher. Biomineralization was induced in simulated body fluid on the bone scaffold’s conductive surface. PPy was also combined with bacterial cellulose (BC) to make conductive hydrogel for the treatment of myocardial infarction. The positive chains of PPy improved the loading of negative charged drug-loaded nanoparticles (NPs). All four systems were successfully developed, fabricated, and loaded with different drugs with different methods to make multifunctional bioengineered systems for drug delivery applications. The results showed that PPy controlled the release of the vancomycin, an antibiotic, and the GATA4-targeted compound 3i-1000, inside the pores of 3D bone scaffold and 3D printed cardiac patch, respectively, which suitable for long-term therapy. Furthermore, bone scaffold, 2D cardiac patch, and conductive BC were combined with microparticles of silica, NPs of porous silicon, and acetylated dextran, respectively, presenting the high potential of in situ drug delivery system enabling and dual delivery of drugs. Overall, this thesis demonstrated that PPy-based biocomposites are successfully developed showing high biocompatibility and high functionality within hard scaffolds and soft patches, and thus, they are very promising platforms for future bone and heart tissue regeneration applications in vivo studies.
  • Reigada, Inés (Helsingin yliopisto, 2022)
    Nosocomial infections imply a great risk for hospitalized patients, causing around 90 000 deaths per year just in the European Union. Bacterial biofilms- microaggregates of bacteria attached to a surface and embedded in a self-produced matrix-are often responsible of this kind of infections, especially those related to medical device use. Medical devices offer an ideal surface for bacteria to attach and form a biofilm and, by switching into this state, bacteria can withstand antibiotic chemotherapy and evade the immune system of the host. One of the current problems to tackle biofilm-associated infections is the limited number of molecules able to act on them at sufficiently low concentrations. For this reason, there is an intense search of biofilm inhibitors, which is often performed via the screening of compound libraries utilizing microplate assays. However, the conditions in which these assays are carried out often differ from those found in clinical environments, and for compounds to be truly effective in translational applications, they ought to be tested in relevant experimental models. This doctoral project has been focused on the development of new in vitro models that better resemble the conditions in which bacterial biofilms are formed and function in medical device-related infections (i.e. orthopedic infections and ventilator-associated pneumonia). Moreover, the anti-biofilm capacity of three previously identified biofilm inhibitors was re-assessed using the newly developed models. These biofilm inhibitors included two dehydroabietic acid derivatives, DHA1 and DHA2 and one flavan derivative, FLA1. In the clinical environment, biofilms can often be composed of multiple species, whose tolerance to antibiotic treatments may be different from those composed of single species. Because of this, in the first study, a co-culture model of Staphylococcus aureus and Pseudomonas aeruginosa was optimized to create a dual species biofilm. These species were chosen because they have been often co-isolated from biofilms related to numerous infections, including medical device-related ones. To be able to develop efficient treatments against multi-species biofilms, it is essential to understand the different biofilm dynamics. Because of this, as part of this study, we exhaustively characterized the variations of the proteomes (specifically the surfaceome and the exoproteome) of S. aureus and P. aeruginosa when co-existing in the same biofilm in comparison to the proteomes of these bacteria in monoculture. Moreover, the hints of several phenotypical changes derived from the proteome analysis were confirmed by a series of follow up studies, which included the analysis of their susceptibility to conventional antibiotics and to the studied compounds (DHA1, DHA2 and FLA1). Out of the three compounds tested, DHA1 was the only one preserving its activity against the S. aureus biofilm formed in co-culture with P. aeruginosa. The second and third studies aimed at assessing the applicability of these three biofilm inhibitors as part of anti-infective materials, specifically bone implanted devices and endotracheal tubes. In the specific case of implantable devices, the prevention of the infection and correct tissue integration are equally important for their success. Therefore, we first developed in the second study a model based on the co-culture of osteogenic cells (SaOS-2), and bacterial cells, specifically, S. aureus, on titanium surface. This model allowed assessing the effects of the newly identified biofilm inhibitors on their anti-biofilm capability on clinically relevant surfaces while simultaneously assessing their effects on tissue integration, apart from providing information on their effects on the interactions between these two cell types. The compound DHA1 was shown to display the best results in terms of prevention of bacterial adherence and cytocompatibility. Nevertheless, when inserting a medical device, not only the cells of the tissue (and possibly bacterial cells) are present, but also immune cells of the host. The insertion procedure often triggers an inflammatory response, which might cause an inefficient clear out of planktonic bacteria due to the efforts of immune cells being directed to degrading the foreign material. Because of this, a third model was developed based on the co-culture of bacterial and host immune cells, specifically neutrophils. This assay aimed at predicting the effects of biofilm inhibitors on the antimicrobial capacity of neutrophils. From the three compounds tested, DHA1 seemed to be the only one increasing the capacity of neutrophils in preventing bacterial adherence to the surfaces. Based upon the results obtained in the three newly developed models, DHA1 was selected to be integrated as part of 3D-printed antimicrobial coating for titanium surfaces. The functionality tests performed on the developed material confirmed its capacity in preventing S. aureus biofilm formation and its cytocompatibility, as well as its activity favoring the antimicrobial capacity of neutrophils. This doctoral research attempts to provide the science community dedicated to anti-biofilm research with new protocols able to shorten the gap between the in vitro testing and the clinical scenario. The results presented here shed some light on the dynamics of a clinically relevant dual-species biofilm, which we hope will contribute to the identification and development of new therapeutic strategies. Finally, it provides novel biofilm inhibitor candidates to be incorporated as part of medical devices, in particular a promising dehydroabietic acid derivative (DHA1) that was successfully incorporated in proof-of-concept studies, within a 3D-printed antimicrobial coating for titanium implants.
  • Stepanova, Polina (2021)
    Parkinson’s disease (PD) and Huntington’s disease (HD) are characterized by loss of function or death of definite cell populations in the basal ganglia. HD is triggered by an expanded polyglutamine tract (glutamine repeats) in the huntingtin protein, leading to misfolding of the protein and subsequent accumulation of mutant huntingtin (mHtt) in the nuclei of different types of neurons. In contrast, in PD, dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) degenerate; moreover, there is also the accumulation of misfolded proteins associated with the neuropathology of PD. Additionally, endoplasmic reticulum (ER) stress has been detected in both of these diseases. Currently, no effective treatment for PD or HD is available to slow, stop, or reverse the progression of neurodegeneration. Cerebral dopamine neurotrophic factor (CDNF) is an evolutionarily conserved protein with neurotrophic properties. CDNF protects and restores the function of DA neurons in preclinical models of PD more effectively than other neurotrophic factors (NTFs), making it a promising drug candidate as a disease-modifying treatment of PD. Additionally, CDNF was safe and well-tolerated, showing therapeutic effects in some PD patients in phase 1/2 clinical trials. This thesis aimed to investigate the potential of CDNF as a drug candidate in cellular and rodent models of both HD and PD. We focused on studying the effect of coadministration of CDNF with another NTF, glial cell line-derived neurotrophic factor (GDNF), in a 6-hydroxydopamine (6-OHDA) rat model of PD. Moreover, we wanted to evaluate the difference in the mode of action between the two factors in this model. We found an additive neurorestorative effect after intrastriatal CDNF and GDNF coadministration in the 6-OHDA toxin model of PD. CDNF alone and in combination with GDNF showed a trend toward an increase in the density of tyrosine hydroxylase (TH)-positive fibers in the lesioned striatal area, and moreover, the combination of the two factors significantly protected TH-positive cells in the SNpc area. CDNF activated the PI3K/AKT pathway, whereas GDNF activated two signaling pathways: PI3K/AKT and MAPK/ERK. We found a delay in activating the prosurvival pathway by CDNF compared to GDNF. Additionally, in comparison with GDNF, CDNF alone significantly enhanced the phosphorylation of ribosomal protein S6 downstream of the PI3K/AKT pathway in lesioned rats. Finally, CDNF, but not GDNF, reduced striatal levels of some ER stress markers in the above model. PD and HD show a wide range of commonalities, and as CDNF has been successful in preclinical trials and demonstrated a positive effect in clinical trials of PD, we expected to observe some beneficial effects of CDNF administration in the experimental HD models. We studied the neuroprotective and neurorestorative potential after different delivery paradigms of CDNF in preclinical models of HD to find the optimal paradigm for possible future application in clinical trials. First, we investigated the protective effects of CDNF in in vitro and in vivo quinolinic acid (QA) toxin models of HD. We demonstrated that CDNF improved motor coordination in QA-lesioned animals, which could be explained by the neuroprotective and neurorestorative effects of CDNF in the cell populations that are vulnerable in HD. Moreover, we found a protective effect after a single intrastriatal injection of CDNF in an in vivo QA-lesion model of HD. Second, we tested the therapeutic efficacy of CDNF in a transgenic mouse model of HD. CDNF was delivered as a chronic intrastriatal infusion using Alzet minipumps for N171-82Q mice. Chronic CDNF administration ameliorated the behavioral deficits and showed a trend toward reduced the nuclear staining and intranuclear inclusions in N171-82Q transgenic mice. Additionally, chronic delivery of CDNF demonstrated a trend toward a decrease in ER stress markers in the striatum in the above model. Furthermore, we found a significant increase in hippocampal BDNF mRNA levels after chronic CDNF administration in N171-82Q mice. In conclusion, CDNF shows a unique beneficial effect in several models of HD independent of the etiology of the disease. Notably, this thesis reports the first beneficial effects of CDNF in different models of HD.
  • Lázaro Ibáñez, Elisa (2017)
    Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies are a heterogeneous population of membrane particles released by cells to the extracellular space and into biofluids during normal physiological and pathological processes. EVs have been recognized as powerful vehicles for intercellular communication due to their capacity to transfer lipids, proteins, and nucleic acids, thereby influencing the properties and functions of recipient cells. Cells generate EVs with a unique composition based on their characteristics, which has a special relevance in the study of diseases such as cancer. Since specific molecular signatures can be passed on to tumor EVs, they are prime candidates for implementation as cancer biomarkers and in the delivery of therapeutics. Thus, exhaustive research is currently targeted towards elucidating the role of EVs in cell-to-cell communication and their therapeutic and diagnostic use. This thesis aims at broadening our understanding of the applicability and functional relevance of the use of EVs as prostate cancer biomarkers and therapeutic delivery vehicles. First, the practical use of EVs as a source of nucleic acid biomarkers in prostate cancer was assessed by exploring the DNA and RNA content of vesicles. Genomic DNA analysis of apoptotic bodies, microvesicles, and exosomes were performed to detect mutations within the EV cargo. The results were validated in plasma EVs of prostate cancer patients, from which the presence of prostate cancer-relevant genes was identified. Next, the prostate cancer-specific messenger RNA signatures of microvesicles and exosomes were analyzed. Unique nucleic acid signatures distinctive for the cell origin were found in the form of differential levels of mRNA transcripts from EV subpopulations. Overall, the nucleic acid content of EVs provided a new source of diagnostic information that could contribute to early prediction and diagnosis of prostate cancer, especially if combined. The role of EV-mediated intercellular communication was shown by comparing the uptake efficiencies and functional effects of EVs from prostate cancer cells of different metastatic status with non-cancer EVs. Additionally, the ability of EVs to carry and deliver a chemotherapeutic drug, together with their cytotoxic effects on prostate cancer cells were also analyzed. While EV uptake, in general, was an active and continuous process, the internalization rate and the subsequent functional effects of EVs on recipient cells differed based on the vesicle origin. EVs derived from cells of a metastatic source were more efficiently internalized than primary prostate cancer or benign prostate epithelial cell-derived EVs. Similarly, those EVs also induced a more proliferative and migratory phenotype in the recipient cells. Applying prostate cancer EVs in the in vitro delivery of paclitaxel to prostate cancer cells, resulted in an enhanced cytotoxic effect of paclitaxel mediated by EV delivery compared to the free drug. In summary, the results presented in this thesis support the concept that EVs can be utilized in both biomarker discovery and drug delivery fields as multifunctional tools for diagnosis and treatment of diseases such as prostate cancer. The studies presented here will also contribute to set the bases for further functional analysis of the roles of EVs in cell-to-cell communication. This new era of research could lead to faster, non-invasive, and more individualized diagnosis and improved treatments tailored to the specific needs of the patients.
  • Raekivi, Pauliina (Helsingin yliopisto, 2021)
    The Finnish medicine reimbursement system is complex and several different conditions required by the Health Insurance Act (1224/2004) and the Social Insurance Institution of Finland (Kela) must be met in order to receive medicine reimbursement. The understanding of medicine reimbursement criteria from the perspective of medicine users has not been studied in Finland before, and little research has been done on the subject internationally. Medicine user-oriented research on the medicine reimbursement system, both in Finland and internationally, has largely focused on the financial opportunities of medicine users to purchase medicines and their opinions on the fairness of medicine reimbursements. The aim of this study was to obtain information on the understanding of medicine reimbursement criteria and the background factors affecting it, the implementation of price, generic substitution and medicine reimbursement counselling in pharmacies, seeking advice on medicine reimbursement, and the financial difficulties of buying prescription medicines. The material used in this study was from the population survey (n=1650), which examined the activation of price competition for pharmaceutical products and customers' expectations of pharmacy operations. The understanding of medicine reimbursement criteria, the price counselling provided in a pharmacy, the effect of financial challenges on the non-buying of medicines and the use of sources of advice related to the medicine reimbursement were described as frequency distributions. The effect of background factors on the understanding of medicine reimbursement criteria was compared using the chi-square test and logistic regression analysis. About a third (31%) of respondents told that they do not understand the basis on which medicine reimbursement is usually received for prescription medicines, and 13% had unclear why they had not been reimbursed for their prescription medicine in the past year. Especially younger age, low income, low medication use, depression and other mental health problems, lack of long-term illness, and insufficient medicine reimbursement counselling in a pharmacy were found to be associated with poorer understanding of medicine reimbursement. 72% of the participants in the study felt that they usually receive sufficient information about the prices of medicines and 61% about the reimbursement of medicines when buying prescription medicines from a pharmacy. Less than half (47%) of respondents felt that they have usually received sufficient information about how the amount of reimbursement for medicines is determined. Slightly over 70% of respondents said that they are usually told about the cheapest medicine available when buying a prescription medicine and/or are suggested to switch to a cheaper one. About 60% were usually told about the difference between the two interchangeable medicines. 88% of respondents would seek information about medicine reimbursement primarily from a pharmacy or pharmacy´s online services. About 3% of all respondents in the study had not bought a medicine prescribed by a doctor for financial reasons in the last six months. Based on the study, about a third of medicine users have remained unclear regarding medicine reimbursements, and not everyone feels that they have received sufficient counselling and information about medicine prices and medicine reimbursements when buying prescription medicines from a pharmacy. Counselling from a pharmacy was found to be related to understanding of medicine reimbursement criteria. Advise on the pricing, medicine reimbursement and generic substitution should continue to be actively provided to medicine users, so that the counselling meets the requirements of the law and the knowledge of the reimbursement system of medicine users can be improved. Based on the results of this study, counselling should be targeted in particular at younger, low-income and from mental health problems suffering medicine users, as well as those who are less familiar with reimbursement issues, for example due to low morbidity or medicine use.
  • Štukelj, Jernej (Helsingin yliopisto, 2021)
    The two parameters used to characterize the effect of a drug in the body are potency and selectivity. Despite their importance, the biopharmaceutical classification system (BCS) and the developability classification system (DCS) are constructed based on permeability and solubility. Furthermore, solubility is not only a molecular property as it is highly affected by the solid-state of a compound. Thus, obtaining a complete understanding of the solid-state impact on the solubility of a drug candidate early in the drug development process is crucial for well-informed decision-making. The overall aim of this thesis was to detect, investigate and quantify the impact of different solid-state forms on the solubility of pharmaceutical compounds using the image-based single-particle analysis (SPA) method. First, the apparent solubility of polymorphs in different solvents was measured. It was demonstrated that the solubility ratio between the two polymorphs is determined by the difference in Gibbs free energy between the polymorphs and it is not affected by changes in pH, ionic-strength and surfactant concentration in dissolution media. Next, the amorphous solubility of a particulate quench-cooled amorphous sample was measured directly. The results correlated well with the two orthogonal methods – standardized supersaturation and precipitation method, and theoretical estimation based on thermal analysis. Moreover, the direct approach of measuring amorphous solubility has proven superior when dealing with highly hygroscopic and fast crystallizing samples. Furthermore, the newly established direct approach of measuring amorphous solubility was then, concurrently with X-ray powder diffraction, used to track two amorphous samples stored at different conditions over an extended period of time. Differences in the crystallization process and its impact on solubility were observed. Machine-vision-detected solid-state changes were demonstrated to provide additional information for the interpretation of the measured pH-solubility profiles of pharmaceutical salts. Based on pH-solubility profiles, values such as pKa, pHmax and Ksp were easily obtained. Moreover, the potential effect of the microenvironmental pH shift was evaluated in the context of solubility measurements. Finally, the solubility of extremely poorly soluble drug – itraconazole – and its co-crystals was measured across a wide pH range and also in biorelevant buffers. A considerable improvement in solubility was detected upon co-crystal formation. Moreover, the solubility of itraconazole in biorelevant buffers was in good agreement with the results in literature obtained previously by the conventional shake-flask method. In summary, all of the above was achieved with less than 100 μg of a sample per measurement. Moreover, non-specificity and the demonstrated broad applicability make the SPA method highly suitable for use in the early stages of the drug discovery and development. The possibility of acquiring a broad level of physicochemical characterization using a single analytical method could significantly accelerate and improve data acquisition leading to enhanced understating of solid-state impact on solubility. Furthermore, with the data quality comparable to the current ‘gold standard’ technique the SPA method could thread the path towards more accurate in silico solubility predictions. Thus, eventually leading to more informed decision-making processes and, finally, optimized and more affordable drug products.
  • Hanzlíková, Martina (Helsingin yliopisto, 2020)
    Gene therapy provides a promising option for treatment of various diseases, but the fact remains that the large number of gene delivery systems has met with little therapeutic success. Viral gene delivery has a high degree of specificity and efficacy, but it does not provide sufficient safety for clinical applications. Therefore, the search for an efficient alternative, a synthetic gene delivery vector, has been active. Typically, non-viral delivery vectors are based on the use of cationic polymers which bind and compact DNA via electrostatic interactions into nanoparticles (polyplexes). The ability of a cationic polymer to bind and condense DNA is important for effective delivery because good packing not only protects DNA against degradation in the extracellular space, but also allows effective release of DNA inside cells. While cationic polymers are relatively nontoxic and safe, they lack significant efficacy. This major drawback of non-viral vectors is largely due to a poor understanding of the mechanism underlying the complexation and gene delivery process. Furthermore, the lack of reliable methods to study the binding between DNA and cationic polymers has hindered development in synthetic gene delivery systems. The aim of this study was to investigate the mechanisms of DNA complex formation and gene transfer mediated by cationic polymers with different structures (poly-L-lysine, PLL; polyethylenimines, PEIs; poly-β-amino esters, PBAEs) and transfection efficiencies. This thesis combines time-resolved fluorescence spectroscopy with cell transfection studies in order to elucidate how polymer structure can affect DNA binding and influence gene delivery outcomes. This method allows the quantitative determination of polymer–DNA interaction and binding. We showed that the mechanism of PEI–DNA and PLL–DNA complex formation was positively cooperative with a saturation limit near 100% at a polymer/DNA molar (N/P) ratio of 2, whereas most of PBAE–DNA complexes expressed negative cooperativity and reached a saturation level close to 80%. The polymer topology, the type of amines (primary, secondary and tertiary) and their density, and the environmental pH had a clear effect on the binding constants and the degree of cooperativity. The possible correlation between fluorescence parameters and transfection efficiency was investigated with a series of PBAEs. Their transfection efficiency showed an increasing trend in association with the relative efficiency of PBAE–DNA nanoparticle formation. The role of free polymer in polyplex formation and gene delivery was examined with PEI as a model vector. For PEI polyplexes, the formation of the polyplex core was completed at N/P 2 and the excess of polymer formed a protective shell around the core. Unlike PLL, PEI molecules were able to undergo an exchange between the core and shell of the polyplexes. Such differences in structural dynamics of these polyplexes may partly provide an explanation for the differences seen in their DNA release and transfection efficacy at the cellular level. The excess of PEI in the shell had no effect on the physical state of polyplexes, suggesting that the polyplex core retains its original structure during shell formation. However, the excess of PEI was a crucial factor in successful transfection. The role of free PEI in the gene transfection process was examined in cell cultures with modified cell-surface glycosaminoglycans content. This study showed that free PEI is essential for minimizing the undesirable binding of polyplexes to cell-surface glycosaminoglycans, which may otherwise pose a barrier in non-viral gene delivery. Lastly, we focused on the role of PEI structure in PEI–liposome–DNA delivery systems (lipopolyplexes). We found that the enhancement of lipopolyplex-mediated delivery by different types of PEI species is common and associated with PEI size rather than structure. In conclusion, the present study demonstrated that the fluorescence spectroscopy approach for the analysis of gene delivery systems can provide valuable quantitative information about the binding behaviour of various cationic polymers to DNA. The improved understanding of mechanisms behind formation of these complexes can contribute to the design of polymeric delivery vectors with improved properties. Furthermore, this study sheds light on the mechanisms by which free polymer enhances gene transfer. It explains why high N/P ratios are needed for effective transfection and how the interactions between free polymer and cell-surface GAGs lead to alterations in gene transfer by the polyplexes.
  • Magarkar, Aniket (Helsingin yliopisto, 2014)
    Drug delivery is a vital issue in pharmaceutical research; once a drug candidate molecule is identified, it must be delivered to the target area of the body where it can take effect. In addition, non-specific distribution of drug molecules to areas other than the drug target must be decreased to avoid unwanted side effects. To achieve this, nanotechnological drug delivery systems can be used. Nanotechnological drug delivery systems come in a wide variety of forms, including liposomes, dendrimers, nanoparticles, and polymeric micelles. Of these, our research is focused on drug delivery liposomes. Drug delivery liposomes are composed of a membrane that forms a closed spherical sack, with a diameter of approximately 100 nm that can contain drug molecules. The criteria for effectiveness of these drug delivery liposomes (DDLs) are structural stability, its lifetime in the bloodstream, the release rate of the encapsulated content and site specific targeting. Cholesterol is one of the crucial lipid components of the DDL known to increase its stability. They also can have a protective polymer coating such as polyethylene glycol (PEG) that protects the DDL from the body s defense mechanisms. Also the DDL can posses targeting moieties, able to direct the PEGylated liposomes to the specific target. In this study we have investigated surface structure of the DDL and its interactions with elements of the blood stream. While it is difficult to determine an accurate picture of the DDL surface and its interactions with ions and bloodstream proteins with atomistic resolution by experiments alone, computational molecular modelling techniques can provide insights into it. Hence, we have used computational modelling and molecular dynamics simulations to understand the role of each component of the DDL in its structure. The three of the five reported studies in this thesis (I, II, III) are focused on how surface charge plays an important role in the liposome, how it is affected by various components of the DDLs, and how the specific interactions of DDLs and ions present in the blood stream influence it. The chapter IV deals with understanding the properties by systematically varying components such as cholesterol and PEG. Also we have produced the first ever model of the first FDA approved drug delivery liposome (DOXIL ®) at atomistic resolution details. The last study (V) deals with the application of molecular dynamics in targeted drug delivery research. In this study we could identify the reason for failure of specific novel targeting peptide (AETP), which is used to functionalize the DDL, by identifying its interactions with the protective PEG polymer.
  • Kolakovic, Ruzica (Helsingin yliopisto, 2013)
    The choice of proper excipients is one of the key factors for successful formulation of pharmaceutical dosage forms. Increasing number of new therapeutic compounds suffers from poor solubility and/or bioavailability, creating a challenge from the drug formulation point of view. Problems have also been encountered in attempts to formulate biological drugs such as peptides and proteins, considering their sensitivity towards certain production processes and routes of administration. In both cases the choice of the right excipient(s) is essential to provide particular processability and development of systems with desirable drug delivery kinetics. The aim of this work was to evaluate pharmaceutical applications of nanofibrillar cellulose (NFC), a renewable, biodegradable and widely available plant based material, as a potential excipient in the production of pharmaceutical dosage forms. Initially, tablets with immediate drug release were manufactured by methods of direct compression and compression after wet granulation using spray dried NFC as a filler material. Addition of NFC improved the flow properties of commercially available and widely used microcrystalline cellulose. The main focus of the thesis was to evaluate NFC material for long-term sustained drug release purposes. This goal was successfully achieved by two approaches: 1) by setting up a spray drying method for the production of drug loaded NFC microparticles, and 2) by developing a simple three-step method for the production of drug loaded NFC films with matrix structures. Both systems were able to sustain the drug release over long periods of time ranging from two months for the spray dried microparticles up to over three months for the films. The drug release kinetics were system and drug dependent, reaching, in several cases, zero order drug release kinetics. The final part of the thesis work focused on studying the interactions between small molecular weight drugs, peptides and proteins with the NFC fibers. The purpose of this study was to further clarify and fully understand the mechanisms behind the successful performance of NFC as drug release controlling material. Binding of drugs to NFC due to the electrostatic interactions was observed. This kind of knowledge is beneficial when choosing the proper drug/excipient combination for the formulation process. In conclusion, NFC was shown to be a versatile excipient for the production of pharmaceutical dosage forms, while the comprehensive evaluation of the full potential of NFC in pharmaceutical applications warrants further experiments in the future.
  • Gatta, Viviana (Helsingin yliopisto, 2020)
    To overcame the spread of bacterial resistance to traditional antbiotics, great interest has arisen towards antivirulence agents, compounds targeting virulence factors. In fact, as there is no link between growth and virulence, antivirulence agents are considered less prone to promote resistance development. In this context, quorum sensing (QS), a communication strategy among bacteria which regulates several bacterial functions including virulence, has been widely investigated for the development of QS inhibitors with the aim of limiting bacterial virulence. This study describes the development of a new assay for the discovery of inhibitors targeting LsrK, a key kinase for autoinducer 2 (AI-2) mediated QS establishment in enteric bacteria. LsrK in fact phosphorylates the AI-2 which, only in the phosphorylated form, can bind to the LsrR repressor and enhance the response to QS signals via activation of the lsr operon. The new assay was used for the screening of three different compound libraries. The best hits from the three campaigns were harpagoside and rosolic acid, presented in Study I, also active in cell-based AI-2 mediated QS assay. Additionally, the active compounds found in Study II and III provided interesting information about the catalytic site of LsrK. To facilitate the confirmation of hits selected by target-based assay and to offer a new tool for the rapid identification of QS inhibitors, Study IV describes the design, optimization and application of a new bioreporter strain, emitting luminescence as response to AI-2 mediated QS activation. The assay was used to test a set of 91 compounds selected to target the ATP binding site of LsrK. The same set of compounds was also tested in the target-based LsrK inhibition assay. The combined results led to the identification of 6 compounds, active in both assays, which thus may decrease response to QS by inhibiting LsrK. Additionally, 18 compounds were active only in the cell-based assay implying that they target other components of the pathway. These findings broaden our knowledge on LsrK and may be used as scaffolds to design compounds with improved properties. Furthermore, the AI-2 mediated QS interference assay represents an additional tool for the identification of QS inhibitors alone or in combination with target-based assays.
  • Hynynen, Marko (Helsingfors universitet, 2017)
    Smoking poses a significant threat to public health. Major public health benefits could be attained if we could increase the incidence of smoking cessation on the national level. However, smoking cessation is often difficult, therefore different medical means have been devised to make it easier for people to quit. One of these medical means is nicotine replacement therapy(NRT). Since NRT entered the market it has been contested which kind of role counselling should have when people purchase NRT. NRT was deregulated In Finland in 2006. Prior to this thesis there has not been carried out studies that would have tried to investigate where the retailer shops are located after deregulation. The aim of this thesis is to investigate how the deregulation affected to the distribution of NRT sales in different NRT-outlets and pharmacies in 2006-2015. It is also aim to investigate if the new outlets have tangibly increased the coverage of NRT-outlets network in diverse municipalities, e.g.in more sparsely populated rural municipalities. Finnish Medical Agency‘s (FIMEA) data about NRT-outlets between the years of 2006-2015 was used as source data for this thesis. This data was analysed by comparing the number of NRToutlets and pharmacies in diverse municipalities. The municipalities have been classified regarding the area and population of diverse municipalities, among other parameters. The PDF sheet of FIMEA’s NRT-outlet data was converted into an Excel file, and using that as a basis different diagrams were made. The number of NRT-outlets grew steadily until during the year 2011 there was a slightly bigger increase in the number of NRT-outlets as NRT became available in restaurants too. Afterwards the growth of NRT-outlets diminished. It can be seen from the results that NRT-outlets, more than pharmacies, are concentrated to the crowded municipalities. On the other hand, there has hardly been an increase in the coverage of NRT-outlet network in more sparsely populated rural municipalities since deregulation took place. However, many of the municipalities that have a pharmacy and one or a few other NRT-outlets are located in Southern Finland surrounding bigger city municipalities or in Central Finland.
  • Liang, Huamin (Helsingin yliopisto, 2013)
    Insufficient delivery of drugs to the target sites like tumors and cells has been a barrier for achieving satisfying therapeutic effects in many diseases. Distribution and exposure of drugs to normal and healthy tissues may enhance the possibility of side effects and toxicity in vivo. Nanoparticle (NP) drug delivery systems have been developed to enable targeting of drugs to target sites and at the same time also reduce or even eliminate the distribution and exposure of drugs to non-targeted sites (normal and healthy tissues). The interactions of ligand attached NPs with specific receptors on the cell surface enable intracellular delivery of drugs. Knowledge of the molecular mechanisms (kinetics and affinity) of specific NP surface interactions is vital for designing and optimizing NPs based targeted drug delivery systems. Biophysical non-labelled surface sensitive detection techniques allow the characterization of the specific NP-cell interactions in vitro at the molecular levels. In this work, surface sensitive non-labelled surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) biosensors were optimized, utilized and further developed as platforms for in vitro characterization and evaluation of the targeting of NP drug delivery systems. A multi-parameter SPR (MP-SPR) prototype was modified, improved and optimized for characterizing molecular surface interactions and phospholipid based thin film properties. The methodologies to extract simultaneously the thickness and the optical properties of thin films were developed by using the multi-wavelength SPR technique. The methodologies were extended to cover the film thickness from few nanometers to micrometers by combining the SPR wavelength and the waveguide mode analysis. These methods were successfully utilized for analyzing LB mono- and multilayers and further for the polyelectrolyte multilayer films. In order to enable the combined use of SPR and QCM techniques for drug and NP interaction studies, these two devices were synchronized to achieve consistent hydrodynamic conditions in the flow channels by computational fluid dynamics (CFD) modelling. The flow channels and the device synchronization were verified by the streptavidin-biotin and liposome-surface interactions. The synchronized SPR and QCM devices were further utilized for the examination of the targeting properties via the streptavidin-biotin liposome interactions under different shear flows. The effect of the flow rate and shear stress on the targeted liposome with the target surface was investigated. The results from SPR and QCM measurements were compared, showing that the binding of the targeted liposome was flow rate and shear stress regulated. According to the SPR measurements, high flow rates improved the binding of liposomes to the target surface. However, the results obtained from the QCM measurements were somehow different. They gave additional information about the liposome binding behavior, indicating deformation or rupture of the bound liposomes at high flow rates and shear stresses. In conclusion, SPR and QCM, the two label free surface sensitive techniques, are excellent platforms for pharmaceutical nanotechnology research. These allow for both the nanoparticle interaction studies and the characterization of nanoscale thin films. Especially, the combined use of the synchronized SPR and QCM techniques forms a powerful platform for the qualitative and quantitative characterization of NP-surface interactions for obtaining in-depth understanding of the targeting behavior of NP drug delivery systems. The results obtained provides the basis for developing new complementary in vitro platforms to traditional cell based in vitro assays for optimizing and screening of NP based targeted drug delivery systems.
  • Hirvonen, Jouni; Salminen, Outi; Vuorensola, Katariina; Katajavuori, Nina; Huhtala, Helena; Atkinson, Jeffrey (2019)
    The Pharmacy Education in Europe (PHARMINE) project studies pharmacy practice and education in the European Union (EU) member states. The work was carried out using an electronic survey sent to chosen pharmacy representatives. The surveys of the individual member states are now being published as reference documents for students and staff interested in research on pharmacy education in the EU, and in mobility. This paper presents the results of the PHARMINE survey on pharmacy practice and education in Finland. Pharmacies have a monopoly on the dispensation of medicines. They can also provide diagnostic services. Proviisori act as pharmacy owners and managers. They follow a five-year (M.Sc. Pharm.) degree course with a six-month traineeship. Farmaseutti, who follow a three-year (B.Sc. Pharm.) degree course (also with a six-month traineeship), can dispense medicines and counsel patients in Finland. The B.Sc. and the first three years of the M.Sc. involve the same course. The current pharmacy curriculum (revised in 2014) is based on five strands: (1) pharmacy as a multidisciplinary science with numerous opportunities in the working life, (2) basics of pharmaceutical sciences, (3) patient and medication, (4) optional studies and selected study paths, and (5) drug development and use. The learning outcomes of the pharmacy graduates include (1) basics of natural sciences: chemistry, physics, technology, biosciences required for all the students (B.Sc. and M.Sc.), (2) medicine and medication: compounding of medicines, holism of medication, pharmacology and biopharmaceutics (side-effects and interactions), patient counseling, efficacy and safety of medicines and medication, (3) comprehensive and supportive interactions of the various disciplines of pharmacy education and research: the role and significance of pharmacy as a discipline in society, the necessary skills and knowledge in scientific thinking and pharmaceutical research, and (4) basics of economics and management, multidisciplinarity, hospital pharmacy, scientific writing skills, management skills. In addition, teaching and learning of "general skills", such as the pharmacist's professional identity and the role in society as a part of the healthcare system, critical and creative thinking, problem-solving skills, personal learning skills and life-long learning, attitude and sense of responsibility, and communication skills are developed in direct association with subject-specific courses. Professional specialization studies in industrial pharmacy, and community and hospital pharmacy are given at the post-graduate level at the University of Helsinki.
  • Silén, Sanna-Mari (Helsingfors universitet, 2012)
    Individually tailored smoking cessation, SC, service provided by community pharmacies is a chargeable special service for customers motivated to quit smoking. The service is based on the PAS service model developed in Great Britain and it has been provided by Finnish community pharmacies since 2006. It includes 4-6 meetings with a specially trained pharmacist, who provides counselling, support, SC plan and follow-up. In this pilot study, the service was investigated from customers' viewpoint, assessing their SC outcomes and experiences. The pilot study was a cooperation project of Division of Social Pharmacy and Association of Finnish Pharmacies. It was a part of a larger SC project co-ordinated by Pulmonary Association Heli and financed by Ministry of Social Affairs and Health. This pilot study assessed the feasibility of the service from customer's viewpoint. It assessed weather the service could increase customers' ability to stay abstinent in different phases of the service. Customers' experiences in relation to SC service and SC itself were also assessed. 14 voluntary pharmacies in different geographical locations in Finland participated in this intervention study and they recruited altogether 36 customers. Before customer recruitment pharmacies received education and introduction of the SC service provided by the Association of Finnish Pharmacies. As part of study protocol, the pharmacies informed local healthcare professionals about the pilot study and asked them to send suitable customers to the service. Pharmacies were paid an expert reward for each customer and they were able to provide SC service to the customers either free or with a low charge. Customers' smoking status and experiences about SC service were assessed with two enquiry forms, which they had filled at the beginning of the service and after three months they had started the service. Their background information was collected with specific background forms during the first meeting and their progress in SC service was investigated by service progress forms. 20 of the 28 customers who returned the first enquiry form and 13 of the 17 customers who returned the second enquiry form were abstinent (55,6 % and 36,1 % of all customers, respectively). All the quitters used some pharmaceutical treatment. Customers who quitted assessed their ability to stay abstinent higher than those who were unable to quit, at the outset and during the service. The customers considered service useful and support of the pharmacist was found important. The customers also considered it significant for pharmacies to provide the SC service. Approximately 32 % of the customers who returned the first enquiry form and 41 % who returned the second enquiry form would pay for the service. They would pay 45 € on average (10-100 €). Multidisciplinary service model was not working as expected, since only a small number of customers were recruited by other healthcare professionals. As a result some of the pharmacies recruited customers also from the pharmacy counter without any contact to other healthcare. 36 % of the 36 customers were abstinent at three months. The control group, planned for the pilot study, failed in recruitment and thus we can only compare our findings to other international studies of the SC service, which have provided similar results. Individually tailored SC service provided by pharmacies is suitable SC support for motivated customers. Customers considered service important and useful, but poor willingness to pay creates challenges for pharmacies to provide this kind of service.