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Prolyl oligopeptidase (PREP) is endopeptidase which cleaves short proline containing peptides. Abnormalities in brain PREP activity has been connected to neurodegenerative diseases. Recently it has been detected that besides its proteolytic activity PREP interacts directly with other proteins which might contribute to generation of neurodegenerative diseases. Further it has been discovered that certain small molecular PREP inhibitors are able to modify these protein-protein interactions (PPIs) and thus have a potential to alleviate the progression of neurodegenerative diseases. This has led to the development of novel second generation PREP ligands which lack the strong inhibitory activity but are potent compounds on modifying the PPIs. Thiazole structure containing PREP modulators has provided most promising class of compounds. It has been detected that these compounds mediate their effects via novel binding site on the enzyme and these effects are not connected to the inhibition of the enzymatic activity. The synthesis of these thiazole containing PREP modulators has proven to be demanding since it have involved a usage of laborious synthesis route and provided low yields. The aim of this research was to examine the synthesis of 2-(2-benzimidazol-1-yl)ethyl)- 4-methyl thiazole containing PREP modulators via previously reported synthesis route. Another aim was to design and develop a synthesis route for 2-(2-(benzimidazol-1- yl)ethyl)-5-bromo-4-methylthiazole, a molecule which serves as valuable intermediate for the lead optimization and generation of second-generation PREP modulators. A synthetic route for 2-(2-(benzimidazol-1-yl)ethyl)-5-bromo-4-methylthiazole was successfully developed. Despite that the total yield of the route remained low. When searching the reasons for the low obtained yield the chemistry behind a thiazole creating cycloaddition reaction and an aromatic halogenation was examined. This led to the discovery of a rare cationic compound which was found to be synthesized from previously undescribed starting materials.

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.

Pharmaceutical companies are currently facing increasing developmental costs, and at the same time, less new compounds are being brought to the market. In vitro -metabolism studies and toxicity assessment of new drug candidates are crucial, as early as possible, to prevent their withdrawal in later development phases. Used study systems are, however, limited and new improved technologies are being investigated. Notable, drug induced liver toxicity and alterations in the liver function are frequent reasons for the drug removals from the development. Human embryonic stem cell (hESC) is one of the most powerful cell types known. hESCs have not only the possibility to divide indefinitely but these cells have also the ability to differentiate to all mature cell types of the human body, such as hepatocytes. This makes them potentially very valuable for pharmaceutical development, in order to create a functional in vitro -model, mimicking the liver tissue. In the literature part, the three dimensional (3D) -hepatic differentiation of mouse and human ESCs in vitro, are discussed. Traditional 2D-culture systems do not adequately mimic the microenvironment of three dimensionally organized native tissue. In 2D-cultures cells grow as a monolayer, when the cell morphology is flattened leading to poor cell-cell and cell-matrix contacts and preventing from the tissue formation. In 3D-culture systems, cells are able to form tissue-like cell integrations, spheroids, and thus, remain their functionality and viability significantly longer. Hydrogels are commonly used biomaterials in 3D-cell cultivation and well known in various areas of tissue engineering for their nano scale porosity and ability to surround cells in 3D-polymer network. In addition, they are capable to absorb large volumes of water and functionalized, in various ways, to improve the required biological or mechanical properties. In the experimental part, the main purpose was to differentiate human hepatic progenitor cells to mature hepatocyte-like cells in three dimensional (3D) -biomaterials. Overall, four different hydrogels (cellulose nanofiber (CNF) hydrogel, HydroMatrixTM, ExtracelTM and PuraMatrixTM) were used as 3D-cell culture scaffolds. Several hepatic cell functions (albumin and urea production and cytochrome P450 (CYP) 3A4 activity) were measured in 2D- and 3D-cultures and compared with the human hepatic carcinoma cells, HepG2, which are often used in drug development. Differentiated hepatocyte-like cells did not show CYP3A4 activity and they produced less albumin and urea compared with HepG2 cells. However, working with hESCs is very demanding and the research in this area is only in the beginning. Therefore, the poor cell functionality results did not come up as a surprise.

Granulation is used to improve the flowability of pharmaceutical powders, reduce the amount of fines and increase the density of the material. Roller compaction has shown growing interest in recent years and it is used ever more frequently in pharmaceutical industry. Roller compaction has many superior qualities compared to wet granulation such as good control of process and absence of moisture and heat in the process. It is also cost effective compared to traditional granulation methods. New APIs are often sensitive to moisture. Therefore traditional granulation methods cannot be used. In the roller compaction process powder mixture is fed between two counter-rotating rolls where the compaction occurs and ribbon is formed. After compaction the ribbon is crushed into granules of desired size. The aim of this study was to find out how the mixture ratio of plastic and brittle material affects the physical properties of roller compacted ribbons such as the strength and stiffness of the ribbons and the structure of the ribbon surface. The materials used were microcrystalline cellulose and dicalsiumphosphate. Nine powder mixtures of 0 to 40 w-% of dicalsiumphosphate were prepared after which the mixtures were roller compacted with the same compactor parameters. Two methods were developed to study the above mentioned characteristics of the ribbons. For the stiffness and strength studies a 3-point bending method was developed for Lloyd material tester. For the surface structure characteristics of ribbons a measurement set up for FlashSizer 3D image analysis device was designed. Bending tests for the ribbons were performed in two different directions. For each batch of ribbons a slope of the linear area and maximum point of bending curves were defined, which represent the stiffness and strength of the ribbons accordingly. Also Young’s modulus and tensile strength were calculated, which are characteristics of a given material. In addition area under curve, which represents the work done to break the ribbon, was calculated. The strength and stiffness of the ribbons decreased with the increasing amount of dicalsiumphosphate. A clear trend was observed. Also Young’s modulus, tensile strength and AUC decreased accordingly. The increase of dicalciumphosphate led to diminished compactibility of the powder mixtures. The compaction force was probably not high enough to fragment the dicalsiumphosphate particles. The ribbons showed higher strength and stiffness when the bending was done perpendicularly ie. across the ribbon width compared to parallel measurements. Also relative standard deviations were smaller in this measurement set up. The 3-point bending method could not mostly distinguish between adjacent formulations from each other but when the difference in the amount of dicalsiumphosphate increased to 10-20 w-% statistically significant differences were observed in most of the calculated values. The surface structure of the ribbons differed between formulations when evaluated visually. Ribbons with less dicalsiumphosphate had a surface structure that followed the knurled pattern of the compactor rolls better.

Many drugs are known to bind to melanin, a complex pigment polymer found in several human tissues. Melanin can act as a natural depot by prolonging the effect of the drug and reducing its toxicity. Since it is highly concentrated in the posterior part of the eye, pigment targeted long-acting drug delivery systems are proposed as an option in ocular diseases. In systemic drug delivery, pigment targeted drugs can potentially distribute to any melanin containing tissue. Therefore, the literature review of the thesis concentrates on the characteristics of melanin and melanosomes, drug binding property and melanin distribution in humans and other species. The main objective of the exploratory part was to determine if melanin binding can be studied with SPECT/CT (single photon emission computed tomography / computed tomography) imaging method. Two different melanin binding drugs, chloroquine and nadolol, were selected and labeled with iodine and radioactive iodine (123I). Equilibrium melanin binding of iodinated and non-iodinated drugs was studied in vitro in order to find out if iodination affects to the binding. Melanin binding was studied in vitro also with non-binding reference salicylic acid, I2-salicylic acid and salbutamol. Finally, melanin binding of 123I-choloroquine and 123I-nadolol was studied with SPECT/CT (NanoSPECT/CT, Bioscan Inc., USA) by comparing distribution kinetics between pigmented and albino rat. Drugs were administered intravenously to the tail vena and the distribution was followed in several time points, up to 24 h. Based on in vitro study, iodination increases melanin binding of hydrophilic drugs, nadolol and salicylic acid, significantly. In vivo study showed clear accumulation of 123I-chloroquine in the posterior eye of pigmented rats whereas it was absent from albino rat. Interestingly, 123I-nadolol accumulated in to the nasal cavity of pigmented rats. Aromatic iodination changes electronegative properties of compounds and raises their logP (octanol/water partition coefficient) value affecting to the melanin binding positively. Therefore the effect of the radiotracer to the physicochemical properties of the compound and melanin binding should be determined in vitro. This study showed that SPECT/CT imaging method can be used to study melanin binding in vivo. Because the method is semi-quantitative, also a quantitative method should be incorporated to the study in order to have more powerful data. Additional studies are required for statistical analysis.

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease affecting motor neurons. It finally leads to the malfunction of the respiratory muscles and death after 1-3 years of diagnosis. Sporadic cases of ALS cover 90-95% of all patients and familial 5-10% respectively. The onset of the disease is usually between age of 40 and 60 and the worldwide incidence is considered to be 1-2/100000. Currently discovered cerebral dopamine neurotrophic factor, CDNF, has showed neuroprotective effects on Parkinson's disease model. What is more, it is known that CDNF is expressed in the muscles of mice and one of its' main functions is to protect cells from ER-stress, one of the pathological mechanisms in ALS. Hence, it is rational to study the effects of CDNF in ALS mouse model. Treatment options are needed, since there is only one approved treatment for ALS, anti-glutaminergic rilutzole. The aim of this study was to find out whether CDNF shows neuroprotective effects in SOD1-mice e.g. by measuring the changes in motor function with different behavioral tests. More over, the distribution of CDNF after intrathecal ventricle injection was studied using immunohistochemical and radioactive labeling methods. The hypothesis was that CDNF is distributed through the cerebrospinal fluid into the spinal cord and muscles in the limbs and shows neuroprotective effects in this SOD1 mouse model.

The leading causes of vision loss in developed countries are related to the impairment of the posterior segment of the eye. The drug delivery to the posterior segment with topical or systemic methods is challenging due to the protective barriers of the eye. The conventional and effective technique to deliver therapeutic concentrations of drugs to the posterior segment is intravitreal injection. Since naked molecules usually have a rapid vitreal clearance, the invasive injections need repeated administration in chronic conditions, resulting to increased risk of complications and poor patient compliance. The growing field of research of drug delivery systems, such as implants, nano- and microparticles and liposomes emphasizes to answer these challenges by enhancing time-controlled and targeted drug release to retinal and choroidal tissues, enabling less frequent administration and reduced off-target side effects. Liposomal drug delivery systems have potential in delivering therapeutics to posterior eye tissues in sustained and targeted manner. The experimental part of the thesis focused on studying the cell uptake, content release and cytotoxicity of light triggered pH-sensitive gold nanoparticle liposomes in human retinal pigment epithelial (ARPE-19), human umbilical vein endothelial (HUVEC) and monkey choroidal endothelial (RF/6A) cell lines. To enhance the cell differentiation to resemble the in vivo morphology, ARPE-19 cells were also used as a filter-cultured model. HUVEC cells were cultured on an artificial basement membrane matrix and induced with vascular endothelial growth factor (VEGF) to form capillary like tube structures. The liposomes were not cytotoxic during 24-hour incubation. All cells internalized liposomes to some extent, but in HUVEC capillary tubes the uptake seemed to be negligible. The light induced calcein release was variable between the experiments, possibly due to the study setting related factors, such as difficulties in temperature control. The liposomal carrier system has promising attributes to posterior eye drug delivery. Liposome-encapsulation prolongs the half-live of a drug. Light triggered release and pH-sensitivity enables highly targeted intracellular drug release decreasing the off-target side effects. Optimization of the study arrangement and liposome production procedure is needed in order to get more reliable results and further assess the future potential of these liposomes in the treatment of posterior eye diseases.

The goal of the thesis was to optimize a dry powder layering process that would produce a swelling polymer layer that could work as a base layer for another layer. The GPCG 1 (Glatt) fluidized bed granulator was equipped with a rotor. Such hydrophilic polymers were used for coating that would not be sensible timewise for wet coating methods because of long process time. For design of experiments Definitive Screening was chosen because it works in situations where time is limited and there is high number of parameters. There were six parameters, four related to the equipment settings and two related to the formulation, that were tested on three levels. The results were used to get optimized parameters using a model in MODDE software. The quality of the coating was analyzed by measuring the friability (strain test with fluidized bed granulator), particle size (dynamic image analyzer), density (helium pycnometer and mercurity porosimeter), erosion (size exclusion chromatography), loss on drying (halogen moisture analyzer) and coating efficiency (weighing and loss on drying). Coating was also analyzed also with scanning electron microscopy. The process was robust with regards to sphericity of the coated pellets. In SEM pictures none of the coatings showed complete film formation. Friability method did not show significant differences between batches. Coating efficiency was high for all batches. Correlations between product characteristics were analyzed and some correlations were observed between including correlations between LOD/CE and densities. No correlation between the densities measured with two different methods were seen. Some of the settings in the DOE were too extreme and produced batches that were very difficult to analyze. Two more batches were produced with adjusted settings. This affected somewhat the ability to develop a reliable model. Model development were also affected by insufficient results from erosion tests and because coating efficiency results with water was used. Some stability problems were noticed during design of experiments and chosen equipment limited the scale of settings. Coating material adhered to the surface of the pellets and process could be adjusted by changing the parameters of the DOE. Some correlations were noticed between formulation, equipment settings and coating properties.

Blood-brain barrier (BBB) is a physical barrier between the blood and the brain. BBB restricts drugs transport from blood stream to the brain, which sets challenges in drug delivery to the brain. Nanoparticles can be utilized in drug delivery to the central nervous system (CNS). Nanoparticles are internalized via endocytosis. However it remains unknown which endocytic pathways are active in brain endothelial cells. The characterization of BBB cells would help light on the exact mechanism of nanoparticle delivery into the brain, which would enable the design of targeted nanoparticles to deliver drugs to the CNS. In present study we characterized human brain endothelial cells, hCMEC/D3, which are widely utilized as BBB in vitro model. As brain endothelial cells are polarized in vivo, the aim of the study was to demonstrate the cell polarization of hCMEC/D3 cells and to study the activity and functionality of different endocytic pathways as a function of cell polarization. The level of cell polarization in cells grown on transwell permeable supports was characterized at multiple timepoints with four different methods: transepithelial electrical resistance (TEER) measurement, lucifer yellow permeability assay, alkaline phosphatase expression and ZO-1 expression. To characterize hCMEC/D3 cells for the presence of specific endocytic pathways, proteins involved into each pathway were selected. Expression of these proteins at mRNA level was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). For clathrin-mediated endocytosis, mRNA level of CHC protein was further correlated with the protein level of this protein, and the activity of clathrin-mediated endocytosis was analyzed by fluorescence activated cell sorting (FACS). Our results showed that hCMEC/D3 cells are best polarized after growing on transwell permeable support for 7 days. At the later timepoints, the cell polarization started to decrease, probably due to multilayer formation. We concluded that measuring TEER alone is not a reliable method to determine polarization status of the cells. mRNA levels of endocytosis-related proteins did not change remarkably as a function of cell polarization. In case of clathrin-mediated endocytosis, there was lack of correlation between CHC mRNA and protein level, but good correlation between mRNA level and activity of the pathway.

Generation of raw materials for dry powder inhalers by different size reduction methods can be expected to influence physical and chemical properties of the powders. This can cause differences in particle size, size distribution, shape, crystalline properties, surface texture and energy. These physical properties of powders influence the behaviour of particles before and after inhalation. Materials with an amorphous surface have different surface energy compared to materials with crystalline surface. This can affect the adhesion and cohesion of particles. Changes in the surface nature of the drug particles results in a change in product performance. By stabilization of the raw materials the amorphous surfaces are converted into crystalline surfaces. The primary aim of the study was to investigate the influence of the surface properties of the inhalation particles on the quality of the product. The quality of the inhalation product is evaluated by measuring the fine particle dose (FPD). FDP is the total dose of particles with aerodynamic diameters smaller than 5,0 µm. The secondary aim of this study was to achieve the target level of the FPD and the stability of the FPD. This study was also used to evaluate the importance of the stabilization of the inhalation powders. The study included manufacturing and analysing drug substance 200 µg/dose inhalation powder batches using non-stabilized or stabilized raw materials. The inhaler formulation consisted of micronized drug substance, lactose <100µm and micronized lactose <10µm. The inhaler device was Easyhaler®. Stabilization of the raw materials was done in different relative humidity, temperature and time. Surface properties of the raw materials were studied by dynamic vapour sorption, scanning electron microscopy and three-point nitrogen adsorption technique. Particle size was studied by laser diffraction particle size analyzer. Aerodynamic particle size distribution from inhalers was measured by new generation impactor. Stabilization of all three raw materials was successful. A clear difference between nonstabilized and stabilized raw materials was achieved for drug substance and lactose <10µm. However for lactose <100µm the difference wasn't as clear as wanted. The surface of the non-stabilized drug substance was more irregular and the particles had more roughness on the surface compared to the stabilized drug substances particles surface. The surface of the stabilized drug particles was more regular and smoother than non-stabilized. Even though a good difference between stabilized and non-stabilized raw materials was achieved, a clear evidence of the effect of the surface properties of the inhalation particles on the quality of the product was not observed. Stabilization of the raw materials didn't lead to a higher FPD. Possible explanations for the unexpected result might be too rough conditions in the stabilization of the drug substance or smaller than wanted difference in the degree of stabilization of the main component of the product <100µm. Despite positive effects on the quality of the product were not seen there appears to be some evidence that stabilized drug substance results in smaller particle size of dry powder inhalers.