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(2018)The bed nucleus of the stria terminalis (BNST) is currently widely studied due to its impact in the anxiety-, stress-, and fearrelated behaviours, as well as in addiction. The BNST is highly heterogeneous brain area constituting of set of subnuclei and a variety of neuron populations, properties of which have only partially been revealed by the earlier research. One of the neuron populations, on which only a very little research has been conducted, is the somatostatin (Sst) expressing neurons, highly abundant in the anterodorsal part of the BNST (adBNST), especially in oval and juxtacapsular nuclei of the BNST. This work aims to elucidate the connectivity of this Sst-neuron population, and their role in the behaviours related to BNST activation, particularly the anxiety-, reward-, and drug withdrawal-related behaviours. To specifically study the somatostatin neuron population in the adBNST, I targeted the neurons using stereotaxic delivery of AAV-vectors encoding a myristylated green fluorescent protein (GFP) for neuronal tracing to Sst-Cre-tdTomato reporter line mice (n=2), and Cre-inducible hM3Dq-DREADDs to Sst-IRES-Cre mice (n=21), with Cre-inducible mCherry fluorescent protein as a control (n=20). The mice were treated with activation-inducing 1.0 mg/kg i.p. clozapine-N-oxide (CNO) 30 min prior to the behavioural tests. To assess acute anxiety-like behaviour, I used the elevated-plus maze paradigm and a modified open field test, in which a novel object is introduced to the arena in the middle of the trial. To study the potential effect on reward-associated behaviours, I used the biased conditioned place preference (CPP) test, and for the withdrawal-linked behaviours, we used a method to precipitate the withdrawal symptoms with naltrexone in subchronically morphine-treated mice (n=9 hM3Dq, n=8 control). The neuronal tracing revealed that the adBNST Sst-neurons project to areas known to partake in stress and fear reactions as well as in autonomic and homeostatic control. Namely, projections were seen in medial and central amygdaloidal nuclei, lateral hypothalamus, periaqueductal grey, ventral pallidum, and parabrachial nucleus. In the elevated-plus maze, the CNO-induced activation of the Sst-neurons did not have any effect on the locomotor activity of the mice between the groups. At the same time, Sst activation did not seem to have any significant effect on the time the mice spent in the open arms, nor in the exploratory activities, like the frequency of the head dips or the stretch-attend postures. In line with these results, no effect on the movement between the groups was observed in the open field test. Similarly, no differences in anxiety-related behaviours, like in the time spent in the centre of the arena or in the number of contacts with the novel object during the last phase of the test, were observed. The CPP test failed to show any meaningful rewarding or aversive properties of CNO-induced activation of the Sst-neurons, while the movement rates of the groups during the conditioning trials were not different in statistically significant way. As for the withdrawal symptoms, all the mice showed the predetermined symptoms, but the test failed to show any differences between the study groups. The neuronal tracing revealed connectivity for the adBNST Sst-neurons with brain regions involved in fear- and anxiety behaviour, social encounters, and autonomic control. In spite of this, the CNO-induced chemogenetic activation of the adBNST Sst-neurons failed to show any significant behavioural effects in the chosen paradigms for anxiety-, and reward-related behaviours, and for withdrawal symptoms. Further research is needed to dissect the Sst-subcircuitry of adBNST, both in order to verify the observed output regions, and to elucidate the role these neurons play in modification of behavioural phenotypes.
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(2013)The aim of this study was to obtain basic knowledge of the applicability of a Büchi Spray dryer B-290 for inhalation particle production and its process parameters effects on particle physicochemical properties. The possibility to anneal the particles where also studied. The greater goal was to provide some information about the solutes' crystallization tendency related to chosen process parameters. Two active pharmaceutical ingredients, salbutamol sulphate and budesonide, where chosen as model substances. Spray drying is a suspended particle processing system which is widely applied and it has been in use from the 1940s. The processed pumpable liquid which contains chosen substances is dispersed into droplets and dried to produce particles that are later collected. Spray dryer is used to process food, biochemical and pharmaceutical substances. In the field of inhalation particle processing, however, it is rather a new technology. This is because of the quality limitations of inhalable particles and the challenges in process optimization. From the many process parameters the concentration of the solid substances, inlet temperature and concentration of organic solvent were chosen as variables for the conducted experiments due to their apparent effects on product quality and especially on solid state. A rudimentary box-annealing system was studied for spray dried substances to verify their solid state transformation tendencies. Salbutamol sulphate was annealed in a box with 65% relative humidity and budesonide in 74 % and 100% relative ethanol activities. Particle size and size distributions were measured with laser diffraction apparatus, crystallinity was analyzed with powder x-ray diffraction and particle morphology was studied with scanning electron microscope. Salbutamol sulphate turned out to be amorphous and budesonide crystalline when spray dried. Both products were within the inhalable size range (1-5µm). Under the current setup the solid state quality of the products was found dependent on the concentration of the solid substances to some extent. Spray dried amorphous salbutamol sulphate was successfully anneaed to a crystalline material and partly crystalline budesonide was annealed to a more crystalline state. Further studies are needed to utilize the full potential spray drying has to offer for inhalation formulating. The kinetics of the annealing procedure and its dependency on the method used still remain largely unexplored.
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(2020)Extracellular vesicles (EVs) are a very heterogeneous group of cell originated nanoparticles that act as mediators of intercellular communication. Accurate characterization of EVs is essential to enable their wider use and development as possible biomarkers, drug carriers, and vaccines. There is no validated reference material with EV-like properties currently available. A validated reference material would improve the reliability and reproducibility of EV studies. Nanoerythrosomes (NanoE) have been studied as a possible option for biological reference material. We aimed to further characterize and compare properties of NanoEs and erythrocyte-derived EVs (EryEV) and assess their stability concerning concentration and size distribution at most commonly applied storage temperatures, +4°C, -20°C, and -80°C for 12 weeks. Characterization was done using nanoparticle tracking analysis and flow cytometry. In addition, we studied the surface protein expression including CD235a, CD47, and CD41 of NanoEs and EryEV and conducted a preliminary cellular uptake test using PC-3 cells, CFSE-labeled NanoE, and EryEV particles. For both, NanoE and EryEV samples, 20°C was the worst storage condition. NanoEs stay stable at +4°C for a month and at -80°C, there were some drops in concentration during the 12 weeks of the experiment. EryEVs stay stable at +4°C and -80°C for 12 weeks. Both NanoE and EryEV particles seemed to be taken into the PC-3 cells, but due to problems with autofluorescence we conclude that confirming studies with different labeling protocols or another method need to be conducted. Both NanoEs and EryEVs samples had a significant number of CD47-positive particles.
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(2018)Formulation development for protein drugs should base on the knowledge of the mechanism of protein degradation. Excipients and formulation can be chosen to stabilize the protein and prevent decomposition. Stability testing is important to identify the likely degradation routes and provide information for formulation development and stability-indicating analytical method development. Gonadotropin-releasing hormone (GnRH) is a neuropeptide hormone that regulates the synthesis and release of gonadotropins: luteinizing hormone (LH) and follicle-stimulating hormone (FHS). Analogs of the endogenous GnRH have been developed to achieve more potent and longer-acting agonists or antagonists. GnRH agonists degrade in several pathways. The primary degradation routes are hydrolysis/backbone cleavage, oxidation, isomerization and aggregation. The stability of GnRH agonists in solid dosage forms has not been studied as excessively as in solutions. The objective of this study was to evaluate the stability of a GnRH agonist (API) at different storage conditions in powder form and in tablet formulations with maize starch or hydroxypropyl methylcellulose (HPMC). The samples were stored for three months at 5 °C (common refrigerator conditions) 25 °C/58 %RH (long-term conditions), and 40 °C/75 %RH (accelerated storage conditions). The samples were analyzed using high performance liquid chromatography. Additionally, the mechanical properties of the formulations and tablets were studied. The stability of API was confirmed in tablet dosage form, when maize starch or HPMC were used as excipients. No degradation products of API were found. As a pure powder API did not degrade either, but it did not stay physically stable at 40 °C/75 %RH. Stressed conditions could be used to find out degradation products in solid state that were not found in this study. Further, the formulations were not ideal, because neither of the studied excipient produced tablets with desirable properties.
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(2012)Improvements in drug screening technology have resulted in a situation where more poorly soluble compounds enter the drug development pipeline. Poor aqueous solubility is a major issue especially in preclinical toxicity testing, where the generation of high drug loads is needed. For oral delivery, liquid formulations are often used and suspensions are potential options for poorly soluble drugs. While several different techniques to enhance solubility exist, most of them have method specific disadvantages or are not universal. Solid state modification, and especially the use of the high energy amorphous form, offers an efficient technique to enhance dissolution properties of a wide range of compounds. A problem of the amorphous form, however, is its physical instability. Amorphous drug in aqueous suspension can re-crystallize via solid-solid and/or solution-mediated pathways. To maintain the solubility advantage of amorphous forms for sufficient period of time, stabilization is needed. One way to stabilize the amorphous form is to prepare a solid dispersion, where the amorphous drug is dispersed in a stabilizing hydrophilic carrier matrix. Another way to add stabilizing agents is to dissolve them into the suspension medium prior to the amorphous solids. Solubilizing polymers may elevate the equilibrium solubility and reduce the driving force for solution mediated crystallization. The aims of this study were to stabilize amorphous indomethacin in aqueous suspensions and to understand the mechanisms behind stabilization. Indomethacin (IND) was used as a poorly soluble model drug (BCS class II). Four different polymers (PVP, HPMC, HPMC-AS and Soluplus®) were selected as stabilizing agents. Crystallization of solid amorphous IND and the concentration of dissolved IND in water were studied after adding: i) the pure amorphous IND, ii) solid dispersions (SDs) at 1:1 and 9:1 drug:polymer ratios (w/w), and iii) the pure amorphous IND into aqueous medium containing predissolved polymer at concentrations of 10 mg/ml or 1 mg/ml, total drug and polymer concentrations being equivalent to 1:1 and 9:1 drug:polymer ratios (w/w) in the SDs, respectively. For HPMC-AS only a 1 mg/ml polymer concentration was used due to its limited solubility. Both the solid and solution phases of the suspension were analysed at different time points for up to 24 h or until crystallization had occurred. Phase transformations in the solid phase were analysed using ATR-FT-IR spectroscopy combined with principal component analysis. The concentration of dissolved drug over the time was assessed by UV spectroscopy. In general, all the polymers, either in SDs or pre-dissolved in medium delayed the onset of crystallization of amorphous IND. Higher polymer concentrations inhibited the crystallization longer than lower ones. A general trend was that SDs were superior in stabilization of amorphous solids, but pre-dissolved polymer solutions generated and maintained higher IND concentrations in solution. Of the four polymers studied, Soluplus® showed the most promising results: SD of Soluplus® and IND at 1:1 ratio (w/w) stayed amorphous in aqueous medium for more than 28 days. On the other hand, crystallization was quite rapid (30 min) when the amount of polymer was inadequate (9:1 w/w). Soluplus® solution (10 mg/ml) generated a 20-fold higher IND concentration than the corresponding SD, possibly due to micellisation. Different polymers showed different abilities to inhibit crystallization and enhance the drug concentration in solution. The addition method and the drug-polymer ratio had an influence on the stabilization abilities of the polymer. Stabilization mechanisms may be both thermodynamic (type of polymer) and kinetic(method of addition).
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Staphylococcus aureus taudinaiheuttajana ja yhteisviljelmämenetelmän käyttö antimikrobiseulonnassa (2010)Staphylococcus aureus is a common commensal and significant opportunistic pathogen. It causes a wide range of infections from superficial skin infections to serious invasive infections. Its pathogenicity is affected by many factors, such as different surface proteins as well as the excretion of toxins and extracellular enzymes. It has many ways to defend a host defense system, such as the formation of capsule and small-colony variants as well as intracellular hiding. Treatment of infections is hindered due to its ability to form resistance to almost every antimicrobial agent used. So far the development of a working and effective vaccine has not been successful. The discovery of new antibacterial agents seems to be still the only efficient way to fight against resistant bacterial strains. However, the development of new antibacterial agents has proved to be difficult. Developing new screening methods is important in order for new drugs to reach the market more effectively and to ensure that new derivatives are more effective and safer. The experimental part of this study aimed at establishing a co-culture of host cells and a pathogen, and to investigate active compounds from primary screen with the established method (Kleymann and Werling 2004). Host cells in the co-culture was HL (Human Lung) cell line and the pathogen was S. aureus (ATCC 25923). Experimental work began by determining bacterial colony-forming units (CFU) and its correlation with absorbance. Based on CFU-determinations the bacterial concentration in the culture media was calculated. Next, the method was optimized and validated. In optimization, statistical parameters S/B-, S/N-values, and Z'-factor were used. Method was optimized regarding cell and bacterial concentrations and incubation time. The method was validated using known antimicrobials. Screening of compounds to be studied was carried out in two stages. All the compounds were first screened in a primary screen. The primary screening method was a standard antibacterial measurement based on turbidometry. Those compounds that were active in the primary screen were investigated in a secondary screen with a co-culture method, but none of the studied compounds showed antimicrobial activity against S. aureus. Therefore we studied the impact of medium that was used in the co-culture method to the activity of the compounds. It was found that the medium had a significant effect on the antibacterial activity of the compounds, the activity was weakened in the presence of the medium. In conclusion, w the established co-culture method is a powerful way to obtain simultaneously information on antibacterial activity as well as cytotoxicity, and it is well suited for further testing of promising compounds.
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(2013)Cholesterol-lowering statins are well-tolerated and effective in prevention of cardiovascular diseases. One of the target groups in treatment of dyslipidaemia is type 2 diabetics, who benefit from reducing cholesterol levels even without a history of cardiovascular disease or high cholesterol levels. Common adverse events associated with statins are myopathy and an increase in liver enzymes. Statins have also been shown to slightly increase the risk of developing type 2 diabetes and increase blood glucose levels. The risk seems to increase with higher doses. Higher doses, however, prevent cardiovascular events more than moderate doses. In this cohort study we assessed the relationship between statins and development of diabetes in a Finnish diabetes prevention study. In addition, we assessed the effects of statins on blood glucose levels. The diabetes risk associated with statins was evaluated by a parametric survival-analysis and the impact on blood glucose levels by a mixture model. The material consisted of the follow-up data from the Diabetes Prevention Study (DPS) from year 1993 to 2009 and along with that statistics on reimbursements for prescription medicines from Kela's reimbursement database. The purpose of DPS was initially to determine if type 2 diabetes can be prevented or delayed by lifestyle intervention. Participants with impaired glucose tolerance were randomly assigned to the intervention group or the control group. The intervention group received intensive and individual counseling on healthy lifestyle while the control group was given general information on health behavior. The study revealed that 36,8 % (n = 182) of the participants had used statins during follow-up. Statin use was more common in the intervention than the control group. Simvastatin was the most used statin and statins had been used for four years on average. Statins were associated with a slight increase in risk of developing diabetes and an increase in fasting plasma glucose and 2 h glucose levels. The risk of diabetes was slighter in the intervention group. Further research is needed to determine the mechanism causing the increase in risk of diabetes, if different statins differ in the extent of risk and if a specific patient group or characteristic is more vulnerable to develop diabetes when exposed to statins. The diabetes risk associated with statins should be taken into account when designing a statin trial. Develoment of diabetes should be a pre-specified endpoint and blood glucose and insulin levels monitored during follow-up.
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(2020)The orexin system is an important regulator of the sleep/wake cycle, and small molecule agonists of the orexin receptors could be beneficial for patients suffering from type 1 narcolepsy. To develop new therapeutics, understanding the interactions between the orexin peptides and their cognate receptors is crucial. The three-dimensional arrangement of the orexin-A peptide complexed to its cognate orexin-2 receptor has so far remained elusive. Here, we identify structurally conserved regions at the predicted binding site and conserved residues of the orexin-A peptide by comparing orexin 2 receptor sequences from nine diverse species as well as with insect allatotropin receptor, a distant relative, and orexin-A sequences from 10 different species. We also visualized the conservation of interaction networks in the orexin 2 receptor binding site by building homology models of the putative orexin receptor of Ciona intestinalis and the allatotropin receptor of Manduca sexta. Structural conservation in the binding site is concentrated on transmembrane segments 2-3-7, in particular the salt bridge between D2.65 and H7.39, and a hydrogen bond network between Q3.32-T2.61-Y7.43. Conservation in orexin-A is concentrated on the C-terminus, while the most conserved individual residues of the peptide are L20, G29, I30, and L31. Applying our conservation results into the analysis of two previously suggested binding modes for orexin-A shows that one of the two demonstrates better mirroring of the conserved residues between the peptide and the binding site. Finally, our data shows that the hydrophobic side of helix 1 of the amphipathic orexin-A should be seriously considered in the analysis of binding modes.
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(2019)Heart failure is a disease of major social and economic impact. The disease is most commonly onset by extensive cardiomyocyte death following a myocardial infarction. Five-year mortality of heart failure is higher than some cancers. Loss of cardiac muscle tissue leads to pathological thickening and fibrosis of the left ventricular wall, which eventually further diminish cardiac function. Cardiomyocytes hardly proliferate, which also exacerbates the problem. Several cell signalling pathways are indicated in pathological reprogramming of the heart and the exact significance of these pathways remains to be demonstrated. Treatment strategies based on renewing cardiac muscle, such as direct injection of stem cells into the myocardium, have failed to reach clinically significant effects on heart failure patients. Direct inhibition of pathological cardiac reprogramming by using small molecule modulators remains as an auspicious strategy to treat heart failure. GATA4, or GATA binding protein 4, is a transcription factor expressed mainly in heart, lung, intestine, gonad and liver tissues, which regulates tissue renewal and cell proliferation by controlling protein transcription. GATA4 binds to GATA sequences in DNA with two zinc finger moieties and enables transcription of target genes. Interactions of GATA4 and several other transcription factors are in central role of guiding heart development, hypertrophy and fibrosis. One of these transcription factors is NKX2-5, which synergistically interacts with GATA4. Inhibition of this interaction in rat myocardial infarction model has been shown to protect against development of heart failure. A screening campaign against the transcriptional synergy of GATA4 and NKX2-5 found potent small molecule inhibitors of this interaction, but these compounds are characterised with stem cell toxicity. The aim of the study was to design and synthesise novel derivatives of GATA4-NKX2-5 synergy inhibitor hit molecule with reduced stem cell toxicity. Modifications on the phenyl ring of the hit molecule were designed, which either increase electron density of the ring or possibly alter the torsional angle between the phenyl and isoxazole ring moieties. Activity of the compounds was studied on a luciferase reporter gene system in COS-1 cells and toxicity was analysed on IMR90 human induced pluripotent stem cell line. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide and lactate dehydrogenase (LDH) assays were selected to measure toxicity on stem cells. Stem cell toxicity of several previously synthesised compounds was assayed in parallel with the novel derivatives. Ten novel derivatives were designed, synthesised and assayed. Four of the new compounds, a mono-ortho-methyl, a di-ortho-methyl, a di-meta-methoxy and cyclohexyl derivatives were found to be equipotent inhibitors of reporter gene activity compared to the hit compound. Additionally, the mono-ortho-methyl, di-ortho-methyl and di-meta-methoxy derivatives were less toxic to stem cells than the hit molecule in the MTT assay. Several other derivatives were found to be less toxic, but also non-active in the luciferase assay. None of the studied compounds exhibited notable necrotic toxicity on stem cells, as examined by the LDH assay. According to this study it may be concluded that substituents of the hit molecule phenyl ring may be altered to decrease stem cell toxicity of the compound. Some of the alterations, most notably substituents in the para-position of the phenyl ring and substitution of the phenyl ring with smaller saturated hydrocarbon rings, diminish the activity of the hit compound. Remarkable toleration of ortho-substitution reinforces the hypothesis of phenyl-isoxazole torsional angle significance for toxicity. On the other hand, addition of two methoxy groups to both meta positions most likely lacks any substantial effect on the torsional angle, which implies another mechanism of toxicity avoidance. Activity and improved safety of the novel inhibitors should be confirmed in animal models before any decisive conclusions on the effects of structural modifications on the hit molecule can be made. In addition, other mechanisms of toxicity should be studied with relevant cell-based assays.
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(2016)Membrane-bound pyrophosphatases (mPPases) are a potential target for drugs against many neglected protozoan diseases, such as malaria, leishmaniasis, toxoplasmosis and trypanosomiasis. New drugs against these diseases are urgently needed, as the clinically used ones are either not effective, suffer from side effects, or resistance against them is developing. The mPPases of these protozoans are genetically conserved, while mammalian DNA does not encode them. A drug development project to find mPPase inhibitors was started, based on mPPase structures solved through X-Ray crystallography. Four hit compounds were identified. The aim of this study was to investigate the binding of these hit compounds at the mPPase binding site, and based on these results, to develop and synthesize novel compounds with higher affinity. A hit compound with an isoxazole ring was chosen as the model compound to be developed further. These novel compounds were evaluated by docking them into the binding site. Eight compounds were chosen to be synthesized and four to be purchased. The Suzuki-Miyaura cross-coupling reaction was used to couple the isoxazole core to different aromatic substituents, producing 3,5-disubstituted isoxazoles. The reactions mostly succeeded, but the yields were uniformly low. Developing the reaction using different solvents and reaction conditions did not produce clear results. Thirteen compounds were tested for activity, including an intermediate product of the synthesis. Two of the compounds showed increased inhibition activity compared to the hit compound, with approximated IC50 values of 10 and 40 μM, respectively. The knowledge gained from these studies can be used to further develop more efficient inhibitors.
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(2023)Antibiotic resistance is a global crisis causing increasing number of infections that cannot be treated with conventional antibiotics. The main reasons for the resistance crisis include overuse and misuse of antibiotics, use in agriculture, and decreased interest of pharmaceutical companies to discover and develop new antibiotics. Apart from mortality, antibiotic resistance causes large economical costs due to longer hospitalizations and more expensive treatments. Bacteria can acquire resistance via multiple pathways. Main path for spread of resistance in bacterial populations is horizontal gene transfer (HGT) in which bacteria receive genetic material that contain resistance genes. Bacteria can acquire new resistance traits via mutations in their genome. The emergence of resistance is a natural feature of bacteria and therefore many bacteria and bacteria can quickly acquire resistance towards novel antibiotics. The resistance properties of potential new antibiotics should be studied already during drug discovery and development. This study determined resistance properties of diazaborine compounds, which have been shown to have antibacterial activity especially against Gram-negative bacteria. The studied properties were mutant prevention concentration (MPC) and spontaneous mutation frequency. MPC measures the antimicrobial compound concentrations in which single-step resistant mutants arise. MPC values can be compared to minimum inhibitory concentration (MIC) values to determine range of mutant selection window (MSW) in which single-step resistant mutants are selectively amplified. Spontaneous mutation frequency is a feature for bacteria in presence of antimicrobials. Spontaneous mutation frequencies demonstrate the proportion of emerged resistant mutants from a bacterial population in each antimicrobial concentration. Four diazaborine compounds were studied with Escherichia coli ATCC25922 and the results were compared with ciprofloxacin. Ciprofloxacin had the lowest MPC at 16xMIC, diazaborine compounds 1, 2 and 3 at 32xMIC and diazaborine compound 4 at 64xMIC. Spontaneous mutation frequency of E. coli was on a normal level with each compound.
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(2021)During co-processing, magnesium stearate can induce surface coating on carrier particles in powders which contain at least one other component in addition to magnesium stearate. Magnesium stearate is sometimes added to powder mixtures as it is known to have beneficial effects on powder characteristics, such as physical and chemical stability, and flowability. In order to fully optimize and control the coating/mixing process, it is necessary to be able to characterize the quality of surface coating. Various methods can be used in determining the coating of powder particles. They can roughly be divided into two different categories: direct and indirect methods. For example, spectrophotometric instruments, which are used to visually express the element distribution on particle surface, are considered direct methods. Indirect methods include methods in which coating parameters are inferred using other properties such as water sorption and powder flowability. Principally direct methods have been used in previous studies to determine the quality of coating. Therefore, the area of interest was especially to study indirect methods and compare them to results obtained using direct methods. Having knowledge of the suitability of indirect methods would be interesting as they might have many benefits compared to direct methods, such as quicker analysis speed and cost-efficiency. The aim of the study was to examine the suitability of direct and indirect methods in studying the surface of powders containing magnesium stearate and active pharmaceutical ingredients (APIs) or lactose, more closely how magnesium stearate was placed on carrier particles as well as the uniformity and the thickness of coating layer. The used methods were selected using literature and own consideration while taking the available equipment into account. The powders containing API (d50 < 10 μm) or lactose (d50 > 80 μm) with magnesium stearate had substantially differing characteristics and thus behaved differently. Therefore, there were differences in the suitability of analytical methods in determining surface of powders. Powders containing lactose and magnesium stearate were able to be examined using direct methods (SEM-EDS and ToF-SIMS) and several indirect methods. Samples with API and magnesium stearate were able to be studied with fewer methods. Validation of the suitability of these methods need more research. However, according to the results from this study, it is probable that surface characterization of studied co-particles can be achieved with direct, but also with indirect methods.
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(2018)Liposomes are nano-sized vesicles in which the aqueous phase is surrounded by lipid-derived bilayer. They are excellent drug vehicles for example in ocular drug delivery because they can, among other things, increase the bioavailability and stability of the drug molecules and reduce their toxicity. Liposomes are known to be safe to use, because they degrade within a certain period of time and they are biocompatible with the cells and tissues of the body. Owing to its structure, the surface of liposomes can also be easily modified and functionalized. Light-activated ICG liposomes allow drug release in a controlled manner at a given time and specific site. Their function is based on a small molecule called indocyanine green (ICG) which, after being exposed to laser light, absorbs light energy and thereby locally elevates the temperature of the lipid bilayer. As a result, the drug inside is released into the surroundings. The blood circulation time of liposomes has often been prolonged by coating the liposomes with polyethylene glycol (PEG). Although PEG is generally regarded as a safe and biocompatible polymer, it has been found to increase immunological reactions and PEG-specific antibodies upon repeated dosing. Conversely, hyaluronic acid (HA), is an endogenous polysaccharide, which is present in abundance for instance in vitreous. Thus, it could serve as a stealth coating material which extends the otherwise short half-life of liposomes. One of the main objectives of this thesis was to find out whether HA could be used to coat liposomes instead of PEG. In order to prepare HA-coated liposomes, one of the lipid bilayer phospholipids, DSPE, had to be first conjugated with HA. For the conjugation, potential synthesis protocols were sought from the literature. Ultimately two different reductive amination-based protocols were tested. Consequently, the protocol in which the conjugation was achieved via the aldehyde group of HA, proved to be working. Thereafter, HA-coated liposomes were prepared by thin film hydration from the newly synthesised conjugate as well as DPPC, DSPC and 18:0 Lyso PC. Calcein was encapsulated in the liposomes. HA-covered liposomes were then compared with uncoated and PEGylated liposomes by examining their phase transition temperatures, ICG absorbances, sizes, polydispersities, and both light and heat-induced drug releases. The aforementioned tests were also conducted when the effects of the HA and ICG doubling were examined and the possibility to manufacture HA liposomes with small size was assessed. HA-liposomes showed similar results as PEG-coated liposomes. In addition, successful extrusion of HA-liposomes through a 30 nm membrane was also demonstrated in the results. Doubling of HA did not significantly affect the results. In contrast, increasing the molar amount of ICG by double caused spontaneous calcein leakage even before any heat or light exposure. Based on these findings, HA could work as a coating material instead of PEG, yet further studies are required for ensuring this conclusion. The other key objective was to evaluate the stability of four different formulations, named as AL, AL18, AL16 and AL14, in storage and biological conditions. Based on the differences in the formulation phospholipid composition, the assumption was that AL would be the most stable of the group and that the stability would decrease so that AL18 and AL16 would be the next most stable and eventually AL14 would be the least stable formulation. As in the previous study, the liposomes were prepared by thin film hydration with calcein being encapsulated inside the liposomes. In the storage stability test, liposomes were stored in HEPES buffer at either 4 °C or at room temperature for one month. In the test conducted in physiological conditions, the liposomes were added either to porcine vitreous or fetal bovine serum (FBS) and the samples were incubated at 37 ºC for five days. Regardless of the experiment, phase transition temperatures as well as light and heat-induced drug releases were initially measured. As the test progressed, calcein release, ICG absorbance, size, and polydispersity were measured at each time point. The initial measurements confirmed the hypothesis about the stability differences of tested formulations. In the storage stability test, all formulations, except AL14, appeared to be stable throughout the study and no apparent differences between the formulations or temperatures were observed. On the other hand, the stability of liposomes stored in biological matrices varied so that the liposomes were more stable in vitreous than in FBS and the stability decreased in both media as expected.
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(2014)Surface plasmon resonance (SPR) is a label free technique to study surface interactions. It is based on photon-plasmon coupling. Laser light is directed through a prism and reflects form a metal surface, often gold. At certain conditions, photons turn into plasmons, which then propagate on the metal surface. The refractive index (RI) of the medium close to the metal surface alters the conditions when plasmons can be generated. By changing the incident angle of the light, photon-plasmon coupling can be matched. Thus, change in the SPR sensogram peak angular position (PAP) indicates change in the RI of the sample. Traditionally, SPR has been used to investigate biomolecule dissociation / association kinetics. Recently, it has gained popularity in living cell sensing. Exosomes are 30-100 nm size lipid bilayer structured vesicles, which are excreted by nearly all cells. They play a role in cell-cell communications. Exosomes carry selected cargo from the cells of origin, including mRNA, miRNA, dsDNA and proteins, and they are directed to specific cells, which internalize them. This initiates responses in the recipient cells. The aim of the study was to harvest exosomes from prostate cancer (LNCaP) cells and use SPR as a novel method to detect exosome internalization by these cells. Adhesion proteins were tested in their efficiency to promote confluent cell monolayer formation on SPR gold substrate sensor surface. Nanoparticle tracking analysis (NTA) showed that exosome purification by ultracentrifugation was successful. It was also found that gold substrate supports confluent LNCaP cell monolayer formation. Adhesion proteins did not shorten the incubation time on gold substrate, but helped the cells remain on the sensor during the SPR experiment. Prostate and platelet exosomes were tested on whether they are internalized by LNCaP cells. Control samples with plain medium and PEI/DNA nanoparticles were used. PEI/DNA particles are nonviral gene delivery vectors, which are known to permeate into cells. The SPR results showed RI increase caused 0.9 ° change in the SPR sensogram with the PEI/DNA sample and no change with the medium sample. Exosomes showed more complex responses, both increasing the PAP approximately 0.1 °. Prostate exosome sensogram returned to baseline after sample rinsing, which did not occur with platelet exosomes. It was concluded that SPR shows a response in cell-exosome interactions, which is most likely because of exosome internalization.
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(2021)Nowadays, targetability studies usually require sample modifications and quite often, examination requires the use of directed light in harmful wavelengths. The surface plasmon resonance (SPR) technique does not need either of those actions. With SPR technology, the targetability of biomolecules can be studied in real-time and without any additional labels. The SPR response is received by measuring the change in surface plasmon resonance conditions due to refractive index changes caused by material interactions in the vicinity of a metal sensor surface. In the present study, the targetability of neonatal Fc receptor (FcRn) was studied by SPR. FcRn-mediated targetability studies were performed against protein A and human colorectal adenocarcinoma (Caco-2) immobilized on SPR sensors. The aim of the study was to confirm the FcRn targetability with bare Fc-fragment and Fc-fragment modified nanoparticles (NPs) designed for oral drug delivery. The NPs consisted of a core porous silicon (PSi) particle, entrapped into a lignin capsule, and finally functionalized with the FcRn-targeting ligand. Results confirmed the binding efficacy of bare Fc-fragment with protein A at pH 6.5, which was the critical pH value for preserving the lignin capsule around the PSi NPs. The cell-based SPR response was significantly higher for FcRn-targeted NPs when compared with non-functionalized NPs. According to these results, FcRn-mediated transcytosis emerges with great potential for oral drug delivery via Fc-functionalized NPs.
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(2023)Purpose: Growth hormone deficiency (GHD) is treated with daily injections of growth hormone (GH). Daily injections may cause burden to patients and to family, that may decrease treatment adherence and may result poor treatment outcome. The aim of this study was to study growth hormone treatment burden in Finland and to identify possible treatment challenges and what preferences caregivers have for growth hormone treatment (GHT). Research methods and data: Anonymous semi-structured survey was conducted in 50 pharmacies across Finland. Potential responders were identified when they came to buy growth hormone product and they were requested to complete the survey with tablet computer. Survey consisted of questions including subject characteristics, treatment background, parent satisfaction to treatment, treatment expectations, decision-making process, compliance and non-compliance reasons, and parent preferences. Survey was conducted between June 2021 and April 2022. Results: Total 79 persons responded to survey. All responders were satisfied with current treatment, 79.5% were very satisfied and 29.5% were quite satisfied. 25.6% responded that they don’t have any challenges with the treatment and 74.4% reported at least one challenge. Most common challenges were injection (35.9%), storage requirements (35.8%) and high price (16.7%). Most common reasons for missing a dose were travel or sleep overs (57.7%), forgot to take medicine (30.8%) and medicine runs out (19.8%). Parents described best and most effective GHT in their own words to be with less frequent dosing (25.6%), storage in room temperature (24.4%) and easy-to use device (23.1%). Conclusions: All caregivers were satisfied with the treatment. However nearly 75% of the responders identified treatment-related challenges. The most frequently reported challenge was the mode of administration (injection). When describing optimal GHT, the wish for less frequent dosing interval was the most often mentioned. Data from this survey can provide support in selection the optimal type of GHT for pediatric and adolescent patients.
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(2022)There are certain characteristics in children’s medication process, such as weight or body surface area-based drug dosing and off-label use of medications, that expose children to medication errors. Small children especially are prone to physical injuries resulting from medication errors. High-alert medications bear a heightened risk of causing significant, even life-threatening harm to a patient when used in error. The aim of this study was to promote children's medication safety by identifying medication errors and contributing factors to errors associated with the use of high-alert medications in pediatric medication process in a hospital environment. The data of this retrospective register study consisted of voluntary medication error reports (HaiPro) made in the pediatric and adolescent units at Helsinki university hospital (HUS). ISMP's (Institute for Safe Medication Practices) list of high-alert medications in acute care settings was used to limit the data. The data was analyzed by using both quantitative and qualitative methods. The aim of the quantitative analysis was to report the frequencies (n) and proportions (%) of high-alert medications and routes of administration and the aim of the qualitative analysis was to identify the types of medication errors and contributing factors in the data. ISMP’s high-alert medications accounted for approximately one-fifth (19.7%) of all medication error reports made in pediatric and adolescent units in 2018–2020. Twelve medications and intravenous route covered approximately 65.0% of all high-alert medications and routes of administration mentioned in the data. Medication errors were mostly identified in medication administration stage (43.3%) and administration errors were often preceded by prescribing errors. Dosing errors (20.5%) and documenting errors (16.8%) were the most common medication error types in the data. Errors associated with dosing and infusion rate were most often involved in severe medication errors. The most frequently identified contributing factors in the data were associated with the work situation and conditions, documenting and information transfer or medications. More detailed risk analysis considering high-alert medications and the intravenous medication process and targeting preventive barriers to identified risk areas are recommended in pediatric and adolescent units in the future. Barriers should be planned to cover the entire medication process. Among different types of medication errors, multiple dosing errors and errors during the programming of infusion rate require special attention in the future.
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(2020)Tiivistelmä/Referat – Abstract Background: Biotin is marketed specifically for its hair and nail growth-promoting effects, and its use has become more common in recent years. High doses of 100 mg biotin have also been used to treat MS. There are no high-dose oral products on the Finnish pharmaceutical market. Biotin 100 mg tablets are not available on the global pharmaceutical market either. The University Pharmacy manufactures 100 mg biotin capsules for hospital use. Manual manufacturing of biotin capsules is a resource-intensive process. The physicotechnical properties of biotin such as crystal properties, flowability, hygroscopicity, true density and compressibility properties have not been previously published in the literature. Objectives: The aim of the thesis work was to investigate whether high-dose biotin tablets can be manufactured as an industrial-scale process. To support product development decision-making, the aim of the master's thesis was also to explore the physicotechnical properties of biotin. The main goal was to develop a method for the direct compression of biotin tablets, but also to study the applicability of the wet granulation method. Methods: The crystal form of the raw materials was examined by X-ray powder diffractometer, particle size and particle size distribution by laser velocimeter, and compression behavior by tabletability tests as well as Heckel analysis. The flowability of the raw materials was studied by bulk and tapped density measurements. The production of biotin tablets was studied with six test batches, two of which were high shear wet granulated and four were direct compression processes. The tablets were subjected to European Pharmacopoeia quality tests such as friability, disintegration, and dissolution tests. Results: The particle size distribution of the biotin grade used in the tablets was wide, with an average particle size of 58 μm. Biotin crystals are flaky in shape. Biotin used was the α-crystalline form and its crystalline form did not change as a result of high shear wet granulation. The flow of the biotin grade was extremely poor. Biotin was not found to be particularly hygroscopic. Biotin is brittle, and when compressed, it forms by fragmenting. Pure biotin cannot be compressed into a stable tablet, as even tablets made with high compression forces will form a lid from which the tablet will easily crumble. Biotin sticks to tablet machine’s punches and causes problems in the ejection phase due to high frictional forces. Test batches of the high shear wet granulation process were successful on both eccentric and rotary tablet machine. Two batches of direct compression tests performed on rotary tablet machines had to be stopped after the powder mass got stuck in tablet machine’s hopper. Biotin tablet’s dissolution was slow for all the manufactured batches, with an average of 63-73 % biotin dissolution at 45 min time point. Conclusions: Main property to be optimized for biotin tablet formulations proved to be mass flowability. High shear wet granulation improved significantly flowability. Weight variance of the tablets in the wet granulation batches was also very small. Biotin’s slow dissolution from the tablets was another significant challenge for all the test batches. Further development of biotin tablets should therefore focus on investigating, which measures accelerate biotin tablet’s dissolution. Product development would particularly benefit from the development of a more efficient, ultra-high performance liquid chromatography method for dose analysis of biotin tablets. Wet granulation test batches should be manufactured at different process parameter levels with different excipients and excipient concentrations. Design of experiments statistical approach should be utilized for these further studies so that factor interactions could be detected, and the manufacturing process and drug product could be efficiently optimized.
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(2011)Chronic heart failure is a major worldwide health problem. It is a complex and severe syndrome caused by different kinds of cardiovascular diseases. Cardiac hypertrophy is frequently caused by hypertension and can lead to abnormality in heart contraction, activation of many neurohumoral mechanism and heart failure. The most important neurohormonal mechanisms of heart failure are activation of sympathetic nervous system and the renin-angiotensin-aldosterone system, insufficiently contracting left ventricle, cardiac remodeling and myocyte loss owing to apoptosis. Antihypertensive drug treatment is often used to prevent or decelerate progression of cardiac hypertrophy. Activation of the renin-angiotensin-aldosterone system plays a major role in heart failure. During the past decades angiotensin converting enzyme inhibitors (ACEIs) have been used as firstline treatment of heart failure. ACEI treatment has been shown to reduce mortality associated with chronic heart failure and improve prognosis of the disease. Angiotensin receptor blockers (ARBs) were expected to replace ACEIs in the treatment of heart failure but for the present they are only an alternative to ACEIs. Beta-blocking agents which reduce activation of sympathetic nervous system have established themself as the second most important treatment of heart failure. Diuretics are widely used as the treatment of heart failure but only aldosterone antagonists has been shown to improve prognosis of the disease. Also digoxin is still used in the treatment of chronic heart failure. In the future renin inhibitors, neutral endopeptidase inhibitors, vasopressin antagonists and molecules that affect inflammatory cytokines could potentially be capable of improving the prognosis of chronic heart failure patients. The major object in the present study was to investigate development of left ventricular hypertrophy induced by abdominal aorta banding in male Wistar rats and prevention of hypertrophy by calcium sensitizer levosimendan and angiotensin II receptor blocker valsartan. Also functionality of abdominal aorta banding as a rodent model of cardiac hypertrophy and heart failure was estimated. Abdominal aorta was constricted above the right renal arteries. That leads to pressure overload and increase in cardiac load. Heart response to pressure overload by hypertrophy in the form of wall thickening. 64 rats were assigned to different groups, each having eight rats. Three of the groups were treated with levosimendan with different daily doses (0,01 mg/kg; 0,10 mg/kg; 1,00 mg/kg) and three of the groups were treated with valsartan with different daily doses (0,10 mg/kg; 1,00 mg/kg; 10,00 mg/kg) via drinking water for eight weeks after the surgery. Sham-operated group underwent the same surgical procedures without constriction of the aorta. All the groups were compared to abdominal aorta banded group without any medical treatment. Cardiovascular parameters such as isovolumic relaxation time (IVRT), left ventricle end-systolic (ESD) and end-diastolic (EDD) dimensions, ejection fraction (EF), fractional shortening (FS), cardiac output (CO), stroke volume (SV), interventricular septum (IVS) and posterior wall (PW) thickness were measured eight weeks after the surgery by using cardiac ultrasound. In the present study levosimendan slightly improved systolic function of the heart. Improvement of the systolic function was seen in a tendency to improve ejection fraction and fractional shortening in abdominal aorta banded rats compared to abdominal aorta banded rats without medical treatment. Neither levosimendan nor valsartan affected diastolic function of heart. Diastolic function was measured by isovolumic relaxation time. Neither levosimendan nor valsartan had significant effect on development of cardiac hypertrophy. Cardiac hypertrophy was estimated by measuring heart weight-to-body weight ratio (HW/BW), left ventricular wall thicknesses and left ventricular internal dimensions in systole and diastole. The present study indicates that outflow constriction by aortic banding is clearly a model of cardiac hypertrophy but not of heart failure.
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(2017)Left ventricular hypertrophy (LVH) is defined as an increase in left ventricular mass. It is initially a coping mechanism by which the heart tries to compensate for the increase in load caused by, for example, hypertension, but it will eventually lead to heart failure. LVH is the result of primarily an increase in cardiac myocyte size, in addition to increased apoptosis and necrosis of cardiac myocytes and fibrosis. Current treatment of LVH is based on a treatment of suspected cause, generally hypertension. Antihypertensive medication has been found to have beneficial effects on LVH. However, antihypertensive drugs can not cure LVH completely, hence other treatment options are needed. To identify new possible drug targets, it is important to increase the inadequate knowledge of the mechanisms and signal transduction pathways mediating LVH. The most relevant stimuli causing hypertrophy are considered to be mechanical stretch, as well as some humoral mediators such as angiotensin II and endothelin 1 (ET-1), to which cardiomyocytes respond through activation of several intracellular signal transduction pathways. As a result, cardiomyocyte gene expression and protein synthesis increase and sarcomeres grow and rearrange, resulting in an increase in cell size. In addition, regulation of calcium, contractile function and energy metabolism of cardiac myocytes change. Numerous intracellular signal mediators interact with each other and can compensate for each other, making it difficult to investigate the significance of individual factors. As important signal mediators are considered to include protein kinase C (PKC) and cardiac transcription factors GATA4 and NKX2-5. In vitro studies of cardiac hypertrophy are usually performed with primary cardiac myocytes isolated from the ventricles of neonatal rats. The H9c2 continuous cell line has been used in some studies as an alternative cell model to reduce the use of laboratory animals. In the experimental part of this thesis, the suitability of H9c2 cells for hypertrophy studies was examined by comparing them to primary cardiac myocytes. In addition, experimental compounds targeted to cardiac transcription factors and PKC were studied by exploring their effects on viability and hypertrophic responses of H9c2 cells and primary cardiac myocytes. The toxicity of the compounds and the effects on cell viability were studied using the lactate dehydrogenase (LDH) assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The hypertrophy responses to cyclic mechanical stretch and ET-1 were primarily assessed by measuring the surface area of cells from fluorescence microscopy images. In addition, the relative expression levels of Nppa and Nppb genes in ET-1 stimulated primary cardiac myocytes were studied by quantitative polymerase chain reaction (qPCR). Both stretching and ET-1 caused an increase in the cell surface area in primary cardiac myocytes but not in H9c2 cells. On this basis, the H9c2 cells respond differently to hypertrophic stimuli than primary cardiac myocytes, and the suitability of H9c2 cell line to hypertrophy studies can therefore be questioned. The compounds targeted to cardiac transcription factors were not cytotoxic at 1-30 µM concentrations, but they also had no significant effect on the hypertrophic responses. In contrast, the PKC compound HMI-1a3 at 30 µM was toxic to primary cardiac myocytes and HMI-1b11 at 30 µM was toxic to H9c2 cells. HMI-1b11 and bryostatin-1 also induced changes in the hypertrophic responses of primary cardiac myocytes, but the significance of these results requires further investigation.
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