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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.

In pharmaceutical sciences the pharmaceutical supply chain is often examined from a quality perspective. As the world is becoming more uncertain due to pandemics and conflicts the societal and political situation where the supply chain operates should be considered. Understanding the big picture helps to consider the cause and effect that lead to medicinal shortages. Effects of these shortages can be seen on every level of the supply chain from the manufacturer to the patient, which is why actors on the supply chain can benefit from understanding the background factors. The aim of the master’s thesis was to examine, whether pharmaceutical field actors could affect realisation of geopolitical risks by preparation and examination that the pharmacotherapy would not be interrupted. Second aim was to bring forward political and societal aspects to pharmaceutical availability which are often side-lined by quality aspects in pharmaceutical context. The study was conducted as a qualitative semi-structured interview between October 2022 and February 2023. Participants (n=11) were recruited via e-mail using representative sampling. Due to recruitment problems, convenience sampling was also used. Questions presented to the interviewees were depending on the group (n=3) they were assigned. Term ”geopolitics” was associated mainly with political and economic factors. Main geopolitical risks for Finland were seen to be small market size and distant location. For Europe, the risks were centralisation of manufacturing (and dependence) to Asia due to economic factors and long disruption-prone supply chain. Transport of pharmaceuticals from Asia to Europe was with sea and air cargo. Inside Europe, transport to Finland was with mainly with lorries utilising ferries. Rail transport was mentioned to be used only on one interview. The transport routes were seen to be staying the same in the future both for Asia-Europe and Europe-Finland. Even though risk management is an important part of functioning of every company, the change in the type of risks requires a new mindset in the pharmaceutical field both from the individual actors as well as international organisations. From risk of strikes and natural disasters we have moved to trade wars, pandemic restrictions, and the strategic acting of industries critical to society. At the same time, the ability/willingness of societies to pay for pharmaceuticals is decreasing, which leads to the manufacturers to find new ways to ensure business.

Ischemic heart disease (IHD) and subsequent heart failure are caused by irreversible loss of contractile cardiomyocytes due to low oxygen supply to the heart. As the leading cause of death worldwide, IHD raises an urgent need for regenerative therapies that prevent or reverse loss of cardiomyocytes. The fetal mammalian heart grows by cardiomyocyte proliferation and utilizes glycolysis as main energy metabolism pathway, until it is introduced to increased oxygen and fatty acid supply at birth. Subsequently, cardiac energy metabolism shifts from glycolysis to β-oxidation of fatty acids and cardiomyocytes exit the mitotic cell cycle. Due to cessation of proliferation the heart can no longer regenerate after ischemic injury and responds to it by introduction of maladaptive pathological processes leading to heart failure. To gain deeper insight on the roles of cardiac metabolism pathways and hypoxia in cell cycle activation, we evaluated the effects of pharmacological metabolic modulation and oxygen supply on cardiomyocyte phenotype and hypoxia response. Furthermore, we studied the changes in the metabolic genotype of cardiomyocytes under alterations of oxygen supply. We utilized quantitative reverse transcription PCR (qRT-PCR) to evaluate the effects of hypoxia and metabolic maturation on the expression of genes involved in hypoxia signaling and metabolism of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Additionally, we investigated the effects of five metabolism-modulating compounds on cell cycle and phenotype of both metabolically matured and unmatured hiPSC-CMs, by utilizing high content analysis. We observed presence of hypoxia signaling as an increase in vascular endothelial growth factor A (VEGFA) expression following 3-hour hypoxic exposure. High expression of succinate dehydrogenase complex flavoprotein subunit A (SDHA) in hiPSC-CMs, which was downregulated at hypoxia, confirmed occurrence of oxidative metabolism induced by metabolic maturation. Surprisingly, metabolic maturation tended to increase proliferation and decrease stress response signaling of hiPSC-CMs. Introduction of the TCA cycle intermediate succinate decreased proliferation of metabolically unmatured hypoxic hiPSC-CMs by 8.2 %. Finally, inhibition of the mevalonate pathway and ketogenesis caused no alterations in hiPSC-CM phenotype or cell cycle, but introduction of the ketone body β-hydroxybutyrate tended to increase proliferation, supporting current evidence that ketogenesis plays a role in cardiomyocyte cell cycle regulation. Our observations suggest that hypoxic hiPSC-CMs can be useful in investigating gene expression and phenotype. Even so, additional methodologies are needed for in-depth evaluation of metabolic reprogramming and its effects on cardiomyocyte phenotype.

Extracellular vesicles are cell-derived vesicles which consist of two lipid layers. Extracellular vesicles involve in intercellular communication, maintaining of homeostase and development of pathophysiological states in human body. Extracellular vesicles are promising biomarkers and drug carriers in future. The aim of this study was to develop a method based on time resolved fluorescence microscopy and autologous extracellular vesicles labelled with environmentally sensitive fluorescent probes for studying the distribution of mitose-inhibitor paclitaxel in prostate cancer cells (PC-3) carried by extracellular vesicles. The efficacy of paclitaxel loaded extracellular vesicles was compared to synthetic liposomes. The two subpopulations of extracellular vesicles, exosome -and microvesicle-enriched, were isolated from the PC-3 cell media by differential ultracentrifugation. The size distribution and particle concentration of extracellular vesicles was determined by nanoparticle tracking analysis. DSPC-Cholesterol liposomes were prepared by reverse-phase evaporation method and the size distribution of the liposomes was determined by dynamic laser diffraction and nanoparticle tracking analysis. Paclitaxel was loaded into the liposomes in hydration phase and into the extracellular vesicles by incubating vesicles and paclitaxel. Unbound paclitaxel was removed from samples by ultracentrifugation. The the dose-dependent sytotoxicity of paclitaxel loaded extracellular vesicles and liposomes was evaluated with Alamar Blue viability assay. The release and distribution of paclitaxel from extracellular vesicles in living PC-3 cells was investigated by confocal microscopy and time-resolved fluorescence microscopy. The exosomes had approximately 50 nm smaller diameter than microvesicles and exosome particle concentrations were significantly higher compared to microvesicles. According to viability assays conducted with wide range of concentrations, paclitaxel loaded in microvesicles were slightly more effective than paclitaxel loaded in exosomes. The time-resolved fluorescence microscopy was useful method for investigating the release and distribution of extracellular vesicle bound paclitaxel, since we succesfully detected changes in Paclitaxel-OregonGreen fluorescence lifetime in different phases of the drug delivery process. With confocal microscopy we detected that paclitaxel loaded extracellular vesicles were already uptaken inside the cells after two hours of incubation and after few hours, paclitaxel was detected in microtubules of PC-3 cells and killed PC-3 cells. Extracellular vesicles may improve the accumulation of paclitaxel into tumor cells thus preventing the side-effects of paclitaxel. Nevertheless, PC-3 cell derived extracellular vesicles have ability to increase the PC-3 cell viability, which limits their potential use as drug carrier due to safety issues. In addition, extracellular vesicles characterization and isolation methods lack standardization and the isolation of exosomes and microvesicles is impossible due to this fact. Extracellular vesicles involvement in physiological and pathophysiological states should be investigated throughoutly and their safety as drug carriers should be examined both in animal and human.