Browsing by Title
Now showing items 505-524 of 626
-
(2022)OATP1B1 is an influx transporter that is predominantly expressed in the liver, and it mediates the uptake of many clinically important endogenous compounds and drugs from portal vein blood into hepatocytes. OATP1B1-mediated uptake affects the rate of hepatic elimination of substrate drugs, directly affecting their plasma concentrations. Some naturally occurring single nucleotide variants (SNVs) in the SLCO1B1 gene encoding OATP1B1 can alter the transport function of the transporter resulting in alterations in pharmacokinetics, efficiency and toxicity of substrate drugs. The aim of this master´s thesis was to examine the effect of four naturally occurring SNVs of the SLCO1B1 gene on transport activity, expression, and localization of the OATP1B1 transporter in vitro. SNVs 170G>A (R57Q), 388A>G (N130D), 452A>G (N151S) and 758G>A (R253Q) were created using site-directed mutagenesis in the SLCO1B1 gene presenting in the pENTR221 plasmid. Recombinante baculoviruses were produced in Sf9 cells using the Bac-to-Bac® Baculovirus Expression System and used to transduce HEK293 cells for the overexpression of OATP1B1 wild type and variant proteins. An uptake assay was used to study the transport activity of the OATP1B1 variants in HEK293 cells. Western blotting was used to study the expression of OATP1B1 proteins in membrane vesicles. Immunofluorescence staining was used to determine the localization of OATP1B1 wild type and variants in HEK293 cells. Transport activity of the OATP1B1 variants R57Q and R253Q was significantly decreased compared to wild type. In contrast, transport activity of the N130D ja N151S variants was not significantly altered. The reasons for the changes in transport activity could not be reliably estimated due to the failure to measure the expression levels of OATP1B1 proteins by Western blotting. However, immunofluorescence microscopy revealed that the localization and expression of the all the studied OATP1B1 in baculovirus transduced HEK293 cells were comparable to the wild type. The results of this master´s thesis indicate that SNVs 170G>A and 758G>A can impair the transport activity and substrate uptake functions of OATP1B1 in vitro. Additional in vitro studies of transport activity, expression and localization of the variants R57Q and R253Q will be required to confirm these results. In the future, the R57Q and R253Q variants should be also studied for their possible clinical significance in pharmacokinetics and pharmacodynamics of substrate drugs, as SNVs 170G>A and 758G>A may increase the exposure and the risk for adverse effects of OATP1B1 substrate drugs.
-
(2013)Cellulose has already been used as an industrial raw material for over a century. However, during recent years the nanostructural features of the naturally occurring biopolymer have been fully investigated and characterized through different processing methods as nanofibrillar cellulose (NFC). This has led to a rapid development of novel cellulose based nanoscale materials and advancements in the field of composite materials. NFC offers interesting specific properties that differ from many other natural and synthetic polymers, such as self-renewable raw materials, semi-crystalline morphology, broad chemical modification capacity, biocompatibility and biodegradability. Biocompatibility and the biomimetic aspects of NFC have enabled the fabrication of nanoporous membranes and scaffolds that can function as medical devices (e.g. tissue engineering, wound healing, novel active implants). In this study, the properties of plant-derived NFC, as potential injectable drug releasing hydrogel "implants" were investigated. Three different sized candidate molecules were selected (123I-NaI, 123I-β-CIT and 99mTc-HSA, from small to large respectively) and investigated with the use of a small animal SPECT/CT molecular imaging device. Study compounds were mixed with the NFC biomaterial and injected into the pelvic region of mice. Drug release was observed for a period of 24 hours and the results were compared to saline/study compound control injections. In addition, 99mTc labeled NFC hydrogels were prepared for dual label tracing to observe the hydrogel positioning during the SPECT/CT acquisitions. For the smaller compounds (123I-NaI, 123I-β-CIT), no differences were found in the drug release or absorption in between the NFC biomaterial and saline injections. However, a clear difference was found with the large compound (99mTc-HSA). In the NFC hydrogel, the rate of release was slower and the distribution of 99mTc-HSA was more concentrated around the area of injection. In addition, the NFC hydrogel did not migrate from, or disintegrate, at the site of injection, suggesting a robust enough structural integrity to withstand normal movement and activity. In conclusion, the labeling of NFC was found to be a reliable and simple method. NFC hydrogels have the potential use as drug releasing medical devices with larger compounds. NFC matrix did not have any controlled release effect on the studied small molecules. Therefore further studies are required for more specific conclusions.
-
(2018)Flowability is an important powder character and, despite decades of research, there are still issues in finding a suitable measurement method. Common challenges are sample size and methodology’s suitability for cohesive powders due to their ability to form vault structures. Powder flowability properties depend strongly on particle features such as size and shape. As particles are in contact with other particles and materials, they receive electric charge and form bonds. In addition to these variables, the gravity and shear stress affect the powder. A combination of all these determine the powder properties such as flowability. Besides the particle properties, process and preservation conditions and especially humidity affects the powder properties significantly. Hence, the powder’s flow behavior varies in different conditions. There are several measurement devices available but none of them is able to yield intrinsic values. Reliability of the measurements presents another challenge as the measured values cannot be directly compared with published literature. Moreover, the flow measurement of cohesive powders is either impossible or extremely difficult with the devices currently available and the sample size needs to be sufficient. Hence, there is a need for new devices, which measure powder flow easily in small-scale. Small sample size is important especially when developing new, expensive drugs since their properties need to be explored in order to develop a new formulation. The aim of the empirical study was to develop a device, which measures particularly the flowability of cohesive powders in small-scale. A ground for the study was a device developed at University of Helsinki, which measures powder flowability by utilizing horizontal movement. In addition, the device breaks the problematic vault formation of cohesive powders by jolts. In the study a cuvette, which utilizes the horizontal movement and measures the powder flow, was developed. Flowability tests were run with five powders – Acetaminophen, Pharmatose 80M, Pharmatose 200M, Emcompress®, Avicel PH-101, Avicel PH-102, Avicel PH-200 and Maize Starch. The results were promising and the device was capable of classifying the powders by their flowabilities but more research is still needed.
-
(2023)Poorly water soluble active pharmaceutical ingredients cause problems for the drug development. Solid state modification offers one approach to overcome the issue. In this study, co-amorphous systems and co-crystals were prepared with indomethacin at molar ratio of 1:1 using nicotinamide as a co-former. Co-amorphous systems were prepared by two different preparation methods: melting the physical mixture and then quench cooling it with liquid nitrogen and dry milling with a ball mill. Co-crystals were prepared by liquid-assisted ball milling. After that, the properties, dissolution, and physical stability of the formed formulations were investigated and compared. The characterisation methods were differential scanning calorimetry, X-ray powder diffraction, Fourier-transform infrared spectroscopy, polarised light microscope and scanning electron microscope. In addition, the solubility and physical stability of the formulations were investigated. Co-amorphous systems were successfully prepared by quench cooling the melt and co-crystals by liquid-assisted ball milling. Dry milling did not induce the formation of co-amorphous systems. In the intrinsic dissolution test, both the co-amorphous system and co-crystal enhanced the dissolution of crystalline indomethacin. When examining the dissolution rate with the paddle apparatus, it was observed that the co-crystal had the highest dissolution rate among both powder and tablet samples. The co-amorphous powder sample floated on the surface of dissolution medium which impeded the dissolution of indomethacin. However, co-amorphous tablet sample showed a higher dissolution rate than crystalline indomethacin. Stability testing (25 °C, 18 %RH) showed that the co-amorphous system recrystallised into a co-crystal after two weeks of storage, while the co-crystal was found to stay stable the whole study period.
-
(2011)Use of natural xanthine derivates in medicine is complicated with their physical properties. Theobromine is poorly soluble while theophylline is highly sensitive to hydration. The aim of this study was to improve bioavailability of xanthines by co-crystallization, theophylline was also cocrystallized with carboxylic acids (capric, citric, glutaric, malenic, malonic, oxalic, stearic, succinic) and HPMC. Co-crystallization was performed by slow evaporation and ball milling. Physical stability was checked by wet granulation and water sorption methods, solubility was measured by intrinsic tablet dissolution. Theobromine formed co-crystal with other xanthines and theophylline interacted with all acids except stearic and HPMC, the latter showed alternative interactions based on hydrogen bonding. Hydration resistance was good in theophylline:succinic acid co-crystal and excellent in complexes containing capric, stearic acids and HPMC. Theophylline:HPMC showed improved solubility. The reported approach can promote use of xanthines and can be recommended for other compounds with similar problems.
-
(2019)Background and objectives: Cells secrete extracellular vesicles (EV) and it has been found that cells communicate via EVs. EVs are liposome-like vesicles. Membrane is consisting of a lipid bilayer and hydrophilic moiety is inside the vesicle. It has been found that EVs carry e.g. nucleic acids, lipids and proteins. The aim of this master thesis was to determine whether EVs can transport non-coding RNA (siRNA) into the central nervous system through the blood-brain barrier. In the literature review, investigated methods which has been used to load siRNA into the EVs and how EVs are transported through the blood-brain barrier. The aim of the experimental part was to produce and isolate EVs and to load FAM-labeled dsDNA and siRNA into EVs by physical methods such as sonication and electroporation. Fluorescence measurements were taken to demonstrate FAM-labeled DNA loading into EVs and the functionality of the siRNA-loaded EVs was measured by measuring the expression level of the gapdh gene. Methods: Extracellular vesicles were produced in ARPE-19 and PC-3 cells. EVs were isolated from the cell culture medium by two-step differential centrifugation (DC) and further purified by gradient centrifugation (GC) by using the OptiPrep™-reagent. OptiPrep™-reagent was purified by Amicon 10kDa filtration tubes. The average particle size and size distribution of the isolated EVs were determined by NTA analysis, protein concentration was measured by colorimetric BCA method and EVs were characterized by Western blot method using HSP70 and CD9 antibodies. EVs were loaded with 21 bp length FAM-labeled dsDNA or siRNA by sonication or electroporation. Free nucleic acid and OptiPrep™-reagent were purified from EVs by the size-exclusion chromatography with Sephacryl (S-300) column. Loading efficient of the EVs were studied by measuring the fluorescence (ex 485 nm, em 520 nm) and qPCR method was used to demonstrate the functionality of the siRNA loaded EVs. In qPCR, the expression level of the gapdh gene was measured in dividing ARPE-19 cells. Results: DC and GC purified ARPE-19 and PC-3 EVs had an average particle size of about 140 nm and were successfully characterized by Western blot method. PC-3 EVs were produced in the bioreactor and the yields were enough for loading experiments. ARPE-19 cells produced only small amounts of EVs in culture flasks. The size-exclusion chromatography was a good method to purification free nucleic acids from EVs. The sonication method did not cause EVs to be degradation under the conditions used. Based on fluorescence measurement, FAM-labeled dsDNA could not be loaded into EVs. The functionality of siRNA-loaded EVs could not be demonstrated in ARPE-19 cell experiments. After electroporation large number of EVs were lost and this method of loading siRNA into EVs did not proved to be suitable. Conclusions: ARPE-19 EVs must be produced in the bioreactor to produce enough EVs for loading experiments. The EV purification protocol should be further optimized since the recovery-% of EVs were low after several purification steps. The size-exclusion chromatography is suitable for the purification of the free siRNA from EVs, but the chromatography method needs further optimization and miniaturization. Loaded EVs should be produced by aseptically or alternatively sterilized prior to ARPE-19 cell assay. Physical loading method, such as sonication, can be scaled to larger scale. Sonication method should be optimized e.g. by experimenting with higher temperatures and longer sonication times. The probe sonicator should be tested instead of the water bath sonicator. According to the literature review, the use of extracellular vesicles as carriers for biomolecule delivery into the central nervous system seems to be promising.
-
(2015)Parkinson's disease is a progressive neurodegenerative movement disorder which is characterized by the death of dopaminergic neurons in the substantia nigra. In addition, other neuropathological features of the disease are intracytoplasmic protein inclusions as well as oxidative and ER stress. Currently there is no cure for Parkinson's disease so there is a need for novel therapies which could stop the disease progression. Because neurotrophic factors can promote the survival of neurons they might be beneficial for the treatment of Parkinson's disease. Cerebral dopamine neurotrophic factor (CDNF) has proven to be neuroprotective and neurorestorative in rodent models of Parkinson's disease. However, the development of new therapies requires relevant disease models. The defects of the current models of Parkinson's disease increases the need for better and more descriptive disease models. The literature review of this thesis presents an overview of ER stress and oxidative stress. Their role in Parkinson's disease 6-OHDA, MPTP, α-synuclein and rotenone models is also reviewed. The experimental part consists of three studies. The aim of the first study was to establish a preformed α-synuclein fibril mouse model of Parkinson's disease. The development of the lesion was studied by testing the motoric skills with balance beam, rotarod, wire hanger and cylinder test. In addition, TH and α-synuclein immunostainings from striatum and substantia nigra sections was performed. In the second study the effect of CDNF on mice behaviour and TH- and α-synuclein-immunoreactivity in the α-synuclein fibril mouse model was examined. The same motoric behaviour tests as in the first study were used. The purpose of the third experimental part was to investigate the effect of CDNF and GDNF on ER stress proteins in 6-OHDA rat model of Parkinson's disease. The levels of ER stress markers GRP78 and phosphorylated eIF2α were analyzed by Western Blot. The results of the studies were promising. In further studies the effect of α-synuclein fibrils on mouse behaviour and TH- and α-synuclein-immunoreactivity could be studied for longer time. The effect of CDNF on α-synuclein aggregation could also be studied further. The expression levels of other ER stress markers could be investigated so it would clarify the effect of CDNF and GDNF on ER stress.
-
(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.
-
(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.
-
(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.
-
(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.
-
(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).
-
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.
-
(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.
-
(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.
-
(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.
-
(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.
-
(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.
-
(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.
-
(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.
Now showing items 505-524 of 626