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  • Palttala, Iida (2010)
    Automated dose dispensing is an increasing field in which medicines are packaged mechanically into small one-dose pouches in portions of two weeks. Suitability of tablets for automated dose dispensing has not been researched systematically earlier. The study was made in collaboration with the dose dispensing unit of Espoonlahti pharmacy. The aim of the study was to define optimal characteristics for an automatically dispensed tablet from a viewpoint of the dose dispensing process to reduce breakings and transitions. Breaking means that tablet crumbles, splits up or breaks up otherwise during mechanical dose dispensing process. Transition means that tablet is dispensed in a wrong dose pouch. Percentually breakings and transitions occur very little, but quantitatively plenty and increasingly when automated dose dispensing is becoming more common. Breakings and transitions cause plenty extra work because of correcting pouches, so their amount should be aimed to reduce. In addition, the aim is to find out matters to enquire from the manufacturers of medicines that would help concluding whether a product is suitable for automated dose dispensing based on written information. Results of the study indicate that to reduce breakings and transitions, an optimal tablet product for dose dispensing is rather small or middle sized, coated, strong and without a breakline and the optimal relative humidity of air in the product room of dose dispensing unit would be around 30 - 40 %. Matters to enquire from the manufacturers of medicines besides size, coating, breaking strength and breakline are stability of the product outside of its original package and light, heat and moisture sensitiveness of the product. Besides breakings and transitions, also stability of a moisture sensitive acetylsalicylic acid product (Disperin 100 mg) was investigated in 25 °C/60 % RH because air humidity in the product room is not adjustable. Duration of the test was four weeks. It is enough since it is the maximum time that tablets are outside their original packages during drug dispensing process before use. Tablets were kept in opened original container (bottle), in closed original container, in cassette of dispensing machine and in two different dose pouches (new material and the one in use). According to the results, cassettes are protecting tablets from moisture as poorly as an opened bottle. Instead, new pouch material protects tablets better than the material in use. Results of Raman spectroscopy measurements indicate no change in acetylsalicylic acid to salicylic acid during four weeks test. Moisture affects to tablets by decreasing breaking strength, which may cause more breakings. Air humidity should be adjusted in product rooms or tablets should be unpacked into cassettes as near operating the machine as possible to prevent breakings. Especially when air humidity is high. Among others, a heat sensitive drug product was researched because of the seaming unit of dose dispensing machine which is radiating heat of about 75 °C to pouches if machine is pulled over in the middle of work. Study was performed with variable temperature XRPD. Results of the study of heat sensitiveness indicate that 75 °C for 60 minutes doesn't induce changes in carbamazepine tablet (Neurotol 200 mg). However, results of the study reveal that researched product did not contain the most heat sensitive form of carbamazepine, so other heat sensitive drug products should be examined to get more information about effects of heat.
  • Palomäki, Emmi (2012)
    3D-imaging is based on combining two or more pictures to form one three-dimensional picture. Most of the methods used provide only surface pictures, but tomography acquires also information about the inside-structure of investigated material. Young's modulus is a method, which has been used for long time to determine toughness hard materials, such as steal. In traditional method a beam-shaped piece is bent. When the size of piece, used force and amount of bending are known, Young's modulus of piece can be calculated. Although the method has traditionally been used to research very hard materials, it has been applied without changes with pharmaceutical materials. It is, however, open to the question whether or not the method is appropriate for those materials. There are also methods to determine Young's modulus based on compressing a tablet or using ultrasound. Determining tablet's toughness with ultimate strength test is complicated because it breaks tablet. For that reason it would be good to find compensatory methods to measure strength of tablet. The aim of the study was to validate Flash Sizer 3D appliance, which is used in 3D-imiging. Another goal was to investigate possible correlations between 3D-imiging, Young's modulus and traditional ultimate strength method. Lastly, the feasibility of Young's modulus as a substitute for traditional ultimate strength measurement in self life studies was investigated. Flash Sizer 3D was validated by measuring particle size distribution of pellets, which were made of microcrystalline cellulose (Cellets). Sizes of the investigated pellets were 100 µm, 200 µm and 500 µm. Also binary mixture of 100 µm and 200 µm was investigated. From microcrystalline cellulose was made tablets and 3D-pictures were taken. Ultimate strength test was made for half of the tablets. Young's modulus was measured from half of the tablets in tableting day, day after that and nine days after tableting. Results show that Flash Sizer 3D is suitable for investigating bigger Cellet. With smaller particles distinguishing of tablets wasn't probably good enough. Still it seems to be quite good method to determine surface roughness of tablet. Young's modulus seems to be very promising as compensating method for traditional ultimate strength measurement. In future in self life studies tablets hardness might be able to investigate by measuring Young's modulus and not measuring ultimate strength. If correlation between Young's modulus and solubility meets the case, Young's modulus might also replace also solubility measurements in self life studies.
  • Salonen, Iiro (2010)
    It was given a task to the Finnish Medicines Agency (Fimea) to prepare a national Medicines Information Strategy. The strategy process can be divided into four stages: 1) the collection and analysis of the information, 2) the determination of the strategy and the vision, 3) the realization and 4) the follow-up stage of the strategy. In the European Union (EU) the High Level Pharmaceutical Forum has drawn up the criteria for the high quality medicines information (MI) and the recommendations to improve the quality and availability of the MI directed to the consumers. The most significant medicines related political actions in Finland in the 21st century are the Medicine policy 2010 -document, the strategies of the National Agency for Medicines and the TIPPA-project. The objective of the Master's thesis was to produce the information to Fimea's MI work. The electric questionnaire was drawn up in the Ministry of Social Affairs and Health. After a pilot test, the questionnaire was sent by email to all national Medicines Authorities in EU (n=27) in November 2009. The purpose of the questionnaire was to find out 1) the significance of the medicine information in the national legislation, 2) the possible MI strategies and 3) the control mechanisms of the medicine information directed to the consumers. The medicines information strategies were found in the United Kingdom (UK), Italy and Germany. Furthermore, the strategy process was unfinished in four countries. In the strategy of the UK 25 concrete actions were presented during a three-year strategy period to improve the quality and availability of the MI and to improve the cooperation between public and private actors. The information and communication technology (ICT) was in the centre of the medicines information offered to the consumers. ICT was utilised by publishing Patient Information Leaflets in Internet and by developing medicines information web pages, digitally patient counseling services and quality certificates. The results of the survey can be utilised as a part of the Fimea's Medicines Information Strategy process. Further studies, for example an analysis of the interest groups, are needed before the preparation of the national strategy. Furthermore, experiences of the implementation of the strategy and the results reached in the UK should be clarified.
  • Hakoinen, Suvi (2014)
    Various cardiovascular diseases cause a significant portion of the direct healthcare costs. For this reason it is important to develop effective treatment strategies. In the treatment of heart failure maintaining healthy lifestyle is an important issue in addition to medical treatment. The aim of this study is to find out whether multidisciplinary interventions in the treatment of heart failure have had better patient outcomes and/or cost savings compared to the standard care and has a certain intervention method proved itself to be more effective than another. The study focuses on the published material in the last ten years, which is compared with prior systematic review about the same topic. The study was conducted as a systematic review. The literature search was performed on Medline (Ovid), CINAHL and Scopus databases. MeSH-terms and other keywords were used in the search. Search was limited to English-language studies. The article selection was made after 06.01.2004 published studies based on the inclusion and exclusion criteria. A quality assessment was made to the selected articles. Only valid articles exceeding 60 % of the maximum points were selected to this review. Literature search found 848 references, of which quality assessment was carried out for 17 articles. 12 articles were selected to this review. Based on statistical significance mortality rate was reduced only rarely. There were lots of heterogeneity in the results for reducing hospitalizations or improving the quality of life. Hospital or clinic-based interventions seemed to be slightly more effective than other intervention methods. There have been no major changes in the intervention methods, only the methods using mailed materials have been left out. Also the outcomes used are similar to those in the past, although validated instruments in different areas of determining the effectiveness have not been developed. This study found no clear evidence about the effectiveness of the treatment programs. Hospital or clinic-based interventions might be more effective than other intervention methods. There is a lot of research about the issue, but high-quality cost-effectiveness studies are lagging. The results from different studies are hard to integrate because the ways to measure vary. The effectiveness of the intervention depends on many factors, and the sheer increase in the knowledge does not seem to be crucial. For assessing the profitability of multidisciplinary interventions there is a need for a high-quality research about the cost-effectiveness of the treatment programs.
  • Huttunen, Sari (2014)
    Long-term use of benzodiazepines is not recommended in the aged. Elimination of these drugs is delayed in the aged, which can prolong drug action and expose users to adverse effects. Long-term benzodiazepine use is associated with many adverse effects, including cognitive impairment and falls. However, there are only few published studies dealing with associations between benzodiazepine concentration and clinical outcomes in the aged in long-term use. The aim of this study was to explore association of residual concentration of temazepam, oxazepam and zopiclone with age, gender, kidney function, drug dose and clinical outcomes, such as self-perceived health and functional abilities in aged patients. The data were collected in Pori City Hospital in July 2004. The patients were between 60-98 years of age (median 81) and the majority (79%) of them were women. Residual drug concentrations were analysed from serum from patients using temazepam (n=14), oxazepam (n=11) or zopiclone (n=28) regularly. Residual oxazepam concentration correlated positively with evening dose (p<0.001) and daily dose (p=0.003). Also oxazepam concentration was higher (p=0.017) in patients who took the last dose later (21:00-02:45) compared to patients with earlier dosing time (15:35-19:00). There was no such association between temazepam or zopiclone and dose or dosing time. This might be explained by the fact that there was more dispersion in the dose and dosing time of oxazepam compared to temazepam and zopiclone. There were no other associations between drug concentrations and demographic variables studied. Concerning associations with clinical outcomes, zopiclone concentration tended to be higher in patients who often felt themselves tired during daytime (p=0.087). Surprisingly, residual serum concentration of oxazepam seemed to be higher in patients who were able to walk and manage their shopping independently. Apart from these findings, residual concentration of temazepam, oxazepam and zopiclone associated poorly with clinical outcomes. These findings do not support routine monitoring of residual benzodiazepine concentrations in aged patients.
  • Parhiala, Minna (2020)
    Suomessa on otettu käyttöön nykyiset lääketurvatoiminnot asteittain vuodesta 2012 lähtien. Merkittävimmät muutokset lainsäädännössä olivat haittavaikutuksen määritelmän muutos sekä erityisen turvallisuuprofiilin lääkkeiden asettaminen lisäseurannan piiriin. Haittavaikutusten spontaani ilmoittaminen on edelleen ensisijainen menetelmä lääkkeen turvallisuustietojen keräämiseksi lääkkeen markkinoille saattamisen jälkeisessä vaiheessa. Vaikka spontaani haittavaikutusilmoitusjärjestelmä on ollut olemassa jo 1960-luvulta, on haittavaikutusten aliraportointi valitettavan yleistä. Tämän tutkielman tavoitteena oli arvioida tietoja, kokemuksia ja asenteita lääkkeiden haittavaikutusten ilmoittamisesta, nykyisestä lääketurvalainsäädännöstä sekä lääkkeiden lisäseurannasta ja mustasta kärkikolmiosta terveydenhuollon ammattilaisten keskuudessa Suomessa. Lääkärit (n = 38), sairaanhoitajat (n = 45), farmaseutit (n = 115) ja proviisorit (n = 36) vastasivat verkkokyselyyn. Aineisto analysoitiin osin aineistolähtöisen ja osin teoriaohjaavan sisällönanalyysin avulla. Tutkimuksen teoriasidonnaisena viitekehyksenä toimi Rogersin diffuusioteoria. Tämä pro gradu -tutkielma paljasti joitain eroja haittavaikutusilmoituksiin liittyvissä teidoissa ja asenteissa terveydenhuollon ammattilaisten välillä. Suurin osa vastaajista koki tietävänsä lääkkeiden lisäseurannasta. Lisäseurannasta ennestään tienneiden joukossa, musta kärkikolmio symboli oli paremmin farmasistien (> 84,5%) tiedossa verrattuna lääkäreihin (45,2%) ja sairaanhoitajiin (32,4%). Lääkäreillä oli enemmän kokemusta, mutta vähemmän tietoa haittavaikutusten ilmoittamisesta kuin muilla terveydenhuollon ammattilaisilla. Suurin osa terveydenhuollon ammattilaisista ei muuttanut työskentelytapojaan lisäseurannassa olevien lääkkeiden kohdalla. Tämä tutkielma tuo esiin sen, että terveydenhuollon ammattilaisilla on riittävät tiedot haittavaikutusilmoitusten tekoon, mutta raportoinnin monimutkaisuuden, huonon saatavuuden ja ilmoitusten tekoon rohkaisemattomuuden takia ilmoituksia tehdään vähän. Tulevaisuudessa tietoisuutta lisäseurannan alaisista lääkkeistä on parannettava sekä raportointiprosessia yksinkertaistettava ja tuotava helpommin saataville.
  • Hiltunen, Anna (2015)
    Periodontitis is a globally significant disease which destroys the attachment tissues and alveolar bone of teeth, eventually leading to tooth loss. Biofilms, the most intrinsic lifestyle of bacteria, play a pivotal role in the occurrence of this disease. Periodontal biofilms can be treated with topically administered chlorhexidine and strain-specific antibiotics. However, these antimicrobials do not offer solutions for periodontal attachment tissue and alveolar bone loss. Some therapeutical alternatives for these conventional treatments have been investigated. In numerous studies, periodontitis is treated successfully (increased attachment and/or alveolar bone levels) with topically and systemically administered bisphosphonates. Furthermore, a topically administered bone graft substitute (bioactive glass) has shown to improve periodontal parameters. In addition, bioactive glass has known antimicrobial and anti-biofilm effects. Moreover, a few bisphosphonates have shown antimicrobial activity against some bacterial strains. Hence, both bisphosphonates and bioactive glass are promising materials for dental applications, also raising interest in their combination. Indeed, it could be hypothesized that this combination product could simultaneously treat both the underlying cause (biofilms) and consequences (alveolar bone and attachment tissue loss) of periodontitis. Open research questions remain for the combination product. Is the anti-biofilm effect enhanced when bioactive glass is combined with bisphosphonates? Moreover, do bisphosphonates have intrinsic anti-biofilm properties? These questions are investigated in this thesis, which is a continuation of a recent doctoral dissertation. In this dissertation, a clodronate-bioactive glass combination product was studied by applying it into periodontal pockets. However, anti-biofilm effects were not assessed. In this thesis, a close examination is carried out on these effects, utilizing relevant biofilm models. The aims of this work were to investigate anti-biofilm effects of bisphosphonates (alendronate, clodronate, etidronate, risedronate and zoledronate) (i) alone, administered as solutions and (ii) combined with bioactive glass S53P4. Optimization of the used assay methods (96-well plate assay, Static Biofilm method) was performed. The anti-biofilm effects of bisphosphonate solutions were screened in the 96-well plate assay using a model organism Staphylococcus aureus Newman and a periodontopathogen Aggregatibacter actinomycetemcomitans ATCC 33384. After this, experiments were conducted with bisphosphonate-bioactive glass combinations. The experiments were performed with a single-specie (A. actinomycetemcomitans ATCC 33384) dental biofilm model based on the Static Biofilm method. The model mimics conditions encountered by periodontal bacteria in the oral cavity. In this part, bisphosphonate particle sizes were measured to determine a suitable control material. In addition to bacterial experiments, pH measurements were carried out to gain an insight to a possible anti-biofilm mechanism. Bisphosphonates administered as stand-alone compounds did not have an effect on either the Gram-positive model organism (S. aureus Newman) or the Gram-negative periodontopathogen (A. actinomycetemcomitans ATCC 33384). In contrast, most combinations of bisphosphonate-bioactive glass revealed a statistically significant increase in anti-biofilm effect on A. actinomycetemcomitans ATCC 33384. The combinations were compared to a control composed of inert glass and bioactive glass. In these assay conditions, the risedronate-bioactive glass-combination was the most effective (significant statistical difference, p < 0.05). Other combinations also reduced biofilms (significant statistical differences, p < 0.05), with the exception of clodronate-bioactive glass, where the change was not statistically significant. The most effective combinations (containing risedronate and etidronate) subjected the biofilms to a period of low pH. Conversely, the least effective combination (clodronate-bioactive glass) rapidly became alkaline, similarly to the control compounds (inert glass and bioactive glass). Thus, anti-biofilm efficacy could be connected to lowered pH. This observation is supported by recent literature where A. actinomycetemcomitans has been deemed highly sensitive to acidity. However, establishing the anti-biofilm rank order of bisphosphonate-bioactive glass combinations would benefit from experiments with equal bisphosphonate particle sizes.
  • Kulmala, Veera (2022)
    Parkinson’s disease (PD) is a progressive neurodegenerative disorder with the neuropathological hallmark of intraneuronal inclusions called Lewy bodies (LB). Accumulation of α-synuclein (α-syn) and cellular components into LBs coincides with degeneration of dopaminergic neurons in the midbrain, substantia nigra. Degeneration of dopaminergic neurons eventually leads to motor dysfunctions. Currently, the treatments for PD are symptomatic. For this reason, new disease-modifying treatments are needed to slow down or prevent the disease progression. Neurotrophic factors (NTFs) have been an interest of research for a couple of decades because of their neuroprotective properties. The main aim of this study was to investigate if brain-derived neurotrophic factor (BDNF) reduces pre-formed fibril (PFF) induced aggregation of α-syn in dopaminergic neurons. PFF-model was used to mimic the accumulation of LBs in neurons, as PFFs induce aggregation of endogenous α-syn in neurons. Additionally, the dose dependence of BDNF was tested. The secondary objective was to investigate the interaction of tropomyosin receptor kinase B (TrkB) signaling pathway and α-syn aggregation using TrkB agonists and antagonists. The cultured dopaminergic neurons isolated from the midbrain of mouse embryos were treated with PFFs on the day in vitro (DIV) 8. BDNF or control treatments were added either 1 hour after the PFF-treatment or on DIV 12. Neurons were fixed on DIV 15 and fluorescent immunohistochemistry was performed. After the detection of fluorescence with automated, high-content imaging, image analysis was done for quantifying dopaminergic neurons, and dopaminergic neurons positive for LB-like aggregates by using unbiased image analysis CellProfilerTM software. Both BDNF and positive control glial cell line-derived neurotrophic factor (GDNF) significantly reduced LB-like aggregates in dopaminergic neurons at both timepoints. GDNF was more effective at both timepoints than BDNF. Both tested doses of BDNF lowered the number of LB-like aggregates, but a more robust effect was seen with the higher dose. The highest tested dose for the TrkB agonists was toxic to the cultured dopaminergic neurons, whereas the lower doses did not affect either the survival or the number of LB-like aggregates. BDNF promoted the survival of the dopaminergic neurons despite the survival-reducing adverse effect of TrkB antagonist K252a. This study provided new information on the effects of exogenously added BDNF on PFF-model with primary neuronal culture. Research on the underlying mechanisms of α-syn aggregation and the protective effects of NTFs can forward the development of new therapies against PD.
  • Åman, Olli (2014)
    Plant cells in plant cell cultures can be used for production of secondary metabolites and recombinant proteins. Producing the desired compounds can be problematic since cells grow slowly, yields can be low and sometimes plant cells do not produce the desired compounds. Yields can be increased by various methods, of which optimisation of growth conditions to favour growth and secondary metabolite biosynthesis is one of various strategies. Light quality is known to have an impact on growth of plants and on accumulation of secondary metabolites. Plants receive information of their environment with photoreceptors, which gives plants ability to alter their morphology and biochemistry to adapt to the prevailing conditions. One of the most important factors involved in controlling morphology and metabolism is activity of bZIP protein HY5, which levels are controlled by degradation by E3 ubiquitin ligase COP1. The photoreceptors are divided to three main groups. A group of Blue/UV-A photoreceptors consists of cryptochromes and phototropins. Phytochromes are photochrome photoreceptors of wavebands of red and far-red. UVR8 photoreceptors are specialized to sense UV-B wavebands. Activated photoreceptors reduce the activity of COP1 individually or inductively. Plant cells contain the same genetic information as intact plants. Object of this study is to investigate effects of different light spectra on plant cell mass pigment accumulation, lipid content and accumulation of secondary metabolites. Additionally, the obtained results can be utilized in designing new artificial light sources to enhance growth and nutritional value of horticultured plants grown under artificial light. VTT's callus cultures established from berries of Rubus (raspberry, cloudberry, arctic bramble) and Vaccinium (lingonberry, bilberry, cranberry) were used in this study. The cell cultures were grown in hormone balanced solid media. For this research Valoya provided four different LED light sources with different spectra, ranging between wavebands 400–800 nm. All berry callus cultures were grown for continuous period of 28–31 days under different light sources. Mass pigments, lipid composition, total phenolic concentration and anthocyanins were analysed from each cell cultures which received different light treatments. Samples were pooled and were by freeze dried and milled. Mass pigments were extracted with acetone and analysis was carried out with UPLC-DAD. Extraction of lipids was carried out with petroleum ether followed with transesterification of glycerolipids and silylation of free fatty acids. The lipid extracts were analysed with GC-MS. Phenolic compounds were extracted with methanol and the extracts were treated with Folin-Ciocalteu's reagent and then analysed with spectrophotometer. Anthocyanins were extracted with acidified methanol and a portion of the extracts were hydrolysed to qualify anthocyanidin moieties of anthocyanins. The extracts and the hydrolysed extract were analysed with UPLC-DAD. Analysis of volatile compounds from each light treated samples was carried out with SPME GC-MS. The obtained results were used to compare concentration differences of the analytes under different light treatments. Correlations between the concentrations of the analytes and different wavebands were possible to establish from the results. Activation of cryptochromes and phytochromes reduced certain lipids that are precursors in LOX-pathway which indicates to increased activity of the pathway. Same wavebands which activated the photoreceptors reduced accumulation of mass pigments, whereas, wavebands of far-red increased the concentrations of mass pigments. In some cases it was observed that small difference in light spectra reduced mass pigment accumulation significantly. The plant cell cultures produced mainly anthocyanins which anthocyanidin moieties were same as in intact plants. Cryptochrome and phytochrome activation increased accumulation of anthocyanins. Yields of anthocyanins can be increased significantly with certain spectra significantly. The effect of light spectra did not have as straightforward effect on total phenolic content. Specie- and linewise differences were observed in light conditions where the highest concentrations of total phenolics were obtained.
  • Jormanainen, Miika (2021)
    Pharmaceutical industry is in a process of adopting new technologies due to the growing interest towards the continuous manufacturing approach. However, while the continuous wet granulation process with twin-screw granulator (TSG) has been studied widely, there has been less focus on subsequent continuous granule drying process. As a result, truly continuous granule drying device has not been available for a long time. However, L.B. Bohle has introduced a horizontal truly continuous fluid bed dryer (CFBD) in form of a perforated belt. In such system the wet granules are transported via vibration along the bed without actual fluidization while the hot air dries the granules. Accordingly, Bohle QbCon-25 fully integrated powder-to-tablet system facilitates the combination of a TSG and CFBD. However, the combination is relatively new and only few studies using these methods are available. Aim of this study was to experimentally evaluate the novel Bohle’s CFBD and elucidate the effect of formulation and process parameters of TSG and CFBD on granule residual water content. Additionally, the granules were characterized by their size distribution and bulk and tapped densities. Granule temperature and residence time in the dryer was determined with CubiSens mini sensors. In total 84 wet granulation trials in design of experiments setup were performed and multiple linear regression (MLR) model was used to investigate the effects of different process parameters on granule loss-on-drying (LoD) response. As a result, formulation microcrystalline cellulose (MCC) amount and all studied process parameters of TSG and CFBD showed significant effect on drying result indicating a robust manufacturing process. The increase in the amount of MCC in the formulation as well as the increase in L/S ratio and line rate in the wet granulation was reflected in a higher residual water content in dried granules. However, with increased drying time, airflow rate and inlet air temperature the granule residual water content decreased. The most influential process parameter affecting the granule residual water content was the used L/S ratio. In contrast, the material acceleration which corresponds to the granule drying time was the most significant process parameter of the CFBD affecting the granule residual water. However, the material acceleration did not only correspond to the material residence time in the dryer, but also to the thickness of the granule bed in the dryer. This indicated that the material acceleration is a critical process parameter of the CFBD. However, the thickness of the granule bed could not be measured in real time in this study. Additionally, at intense drying conditions granules showed a very dry outcome and further studies are required to elucidate the operational limit of the CFBD device. Furthermore, the vibration during the drying phase did not have an effect on granule size or the bulk and tapped densities. Material behavior in system was plug-flow like and mean material residence time in the CFBD was only 40 seconds with the middle material acceleration setting. Moreover, the temperature of the granules rose close to the process air temperature, but only for a very short time. Additionally, the used process settings in wet granulation affected the granule temperature, however, the most influential factor on the granule temperature was the used inlet air temperature. Overall, Bohle QbCon25 manufacturing system with TSG and CFBD showed high suitability to wet granulate and dry the produced granules with a uniform residual water content up to 20 kg/h throughput rate without process instability issues.
  • Hämäläinen, Noora (2021)
    Mini-tablets are 1-3 mm in diameter and administered as a single tablet or as a multi-particulate formulation. Mini-tablets are an attractive alternative for conventional solid dosage forms due to the ease of administration and the possibility for combination and individualised drug therapy. In mini-tablet production, good flowability of the formulation is critical as minor variations in die filling can lead to significant changes in mini-tablet weights. In addition, to reduce weight variation, the particle size should not exceed 1/3rd of the die diameter. This study aimed to determine the influence of the granule size on mini-tablet weight variability and content uniformity. The feasibility of direct compression, as well as high-shear wet granulated and roller-compacted formulations, were evaluated. From the nine final formulations manufactured, particle size distribution, Hausner ratio, Carr’s index, angle of repose and flowability were determined. The mini-tablets were made on a rotary tablet press using single punches of 3 mm in diameter. Content uniformity and weight variation of the mini-tablets were determined. The direct compression formulation had the smallest particle size, and the roller-compacted formulation milled through a 1.0 mm and 1.25 mm square screen had the largest particle size. Surprisingly, the RC 0.8 mm grater screen formulation had a very wide particle size distribution and is classified as a very fine blend. The wide particle size distribution might result from a high fill ratio during the milling of the roller-compacted ribbons. The four different high-shear wet granule formulations had a very similar particle size distribution. According to the Hausner ratio and Carr’s index values, the flow properties of the formulations varied between fair and very poor, while according to the angle of repose, the flow properties were between excellent and poor. However, all nine formulations were used to make mini-tablets with acceptable uniformity of mass, mini-tablets were within ± 8 % of the target weight, and none exceeded the 10 % limit set by Ph. Eur. The weight variation is small, as indicated by the low RSD of 1.0-2.9 %. The differences in the weight variation may be attributed to segregation due to particle or granule size and density. This is further supported by the fact that no force feeder or vacuum was utilised in the rotary tablet press, possibly causing re-circulation of the formulation and shearing forces. In addition, the fill level of the feeder might have varied between the nine formulations and affect the weight variation in a way that is not recognised in this study. Only direct compression formulation was within the limits of uniformity of content of single-dose preparations set by Ph. Eur. In the final formulations, the amount of paracetamol was in the HSWG 0.8 mm round screen 98.5 % and in the RC 1.0 mm square screen 97.7 %. These results suggest that the formulations contained an adequate amount of paracetamol, which does not explain why the mini-tablets made from high-shear wet granules did not meet the content uniformity criteria. Furthermore, the weight variation might not entirely explain why high-shear wet granulated formulations performed so poorly in the content uniformity analysis. In summary, that direct compression is a feasible manufacturing method for mini-tablets of 3 mm in diameter. However, further studies are needed on the content uniformity of mini-tablets made using high-shear wet granulated and roller-compacted formulations as these did not meet the content uniformity criterion. In particular, the content uniformity of the mini-tablets made from the high-shear wet granulated formulations was not acceptable, and the reason for this was not identified.
  • Ignatius, Adele (2021)
    Misfolding and aggregation of alpha-synuclein (α-syn) protein, leading to dysfunctional proteins and toxic protein aggregates, are seen as major factors in the pathogenesis of Parkinson’s disease (PD). Direct protein-protein interactions (PPI) between α-syn and a serine endopeptidase, prolyl oligopeptidase (PREP), have been shown to increase α-syn aggregation. Small molecular PREP inhibitors, in turn, have been shown to reduce the ɑ-syn aggregation process both in vitro and in vivo. Inhibition of PREP has been shown to have dual effects on ɑ-syn aggregation: first of all, blocking PREP mediated seeding and secondly, inducing the clearance of ɑ-syn aggregates via increased autophagy. Thus, PREP inhibitors should be further studied as a potential treatment for PD and other synucleinopathies. In this study, we evaluated the effect of two different PREP inhibitors, 4-phenylbutanoyl-L-prolyl-2(S)-cyanopyrrolidine (KYP-2047) and HUP-115 in a virus vector-based unilateral A53T-ɑ-syn overexpression mouse model. AAV-A53T-ɑ-syn injections used in this study caused a significant increase in oligomer-specific alpha-synuclein (ɑ-synO5) immunoreactivity and a mild dopaminergic neuron loss, together with mild motor deficits. Neither 2-week PREP inhibition with KYP-2047 or 4-week PREP inhibition with HUP-115 reduced ɑ-synO5 immunoreactivity or protected dopaminergic neurons in the substantia nigra (SN). Concordant to this, the treatments did not restore the slight behavioral deficit AAV-A53T-ɑ-syn injections caused in the cylinder test. In previous studies, PREP inhibition with KYP-2047 decreased ɑ-synO5 immunoreactivity, attenuated dopaminergic neuron loss and restored behavioral deficits in other α-syn overexpression mouse models. It is suggested that PREP inhibitors mainly have an effect on soluble ɑ-syn oligomers, rather than insoluble fibrils. In case A53T-ɑ-syn forms insoluble fibrils too rapidly in mice, overexpression of A53T-ɑ-syn might not be a suitable option when studying the effects of PREP inhibitors. Our results suggest that further characterization of this model in mice is much needed before drawing any conclusions about the effect of these PREP inhibitors.
  • Jaskari, Iida (2022)
    Multiple sclerosis is a progressive inflammatory disease of the central nervous system that affects young adults. The pathological hallmark of MS is the degradation and loss of oligodendrocytes resulting in demyelination. Damage to axons caused by demyelination severely impairs physical function. Currently there is no cure for MS, but current drugs aim to modify the course of the disease and relieve symptoms. However, they are unable to promote the repair of damaged myelin sheaths, and thus new therapies are needed. In this study, the effect of V-MANF on remyelination was investigated in two commonly used experimental toxin models. V-MANF is a modification of the endoplasmic reticulum located protein MANF, which has been found to have neuroprotective and regenerative properties. Additionally, MANF can regulate ER stress, which contributes to demyelination in MS. The effect of V-MANF on lysolecithin-induced demyelination was examined in organotypic cerebellar brain sections from C57B/6 mice. The study was conducted exceptionally using the brains of adult mice because they are a better model for neurodegenerative diseases. However, when analyzing the results, it was found that there was no demyelination in the tissue cultures, so the effect of V-MANF could not be analyzed. In the other study, C57B/6 mice were given dietary cuprizone for six weeks, followed by daily intranasal administration of either V-MANF or vehicle for seven days. Mice were subjected to behavioral experiments, in which a light/dark box test showed that V-MANFs had a potential anxiolytic effect in mice receiving cuprizone. No significant demyelination was observed by immunohistochemical analysis and therefore the effect of V-MANF on remyelination could not be assessed. However, the results of the study can be utilized in the design of further studies.
  • Niittymäki, Erika (2021)
    Since the discovery of ketamine’s antidepressant response, numerous of studies have been observed it to alleviate depressive symptoms rapidly and effectively within hours. This is a significant advantage compared to traditional antidepressants, which take weeks to show treatment efficacy. Ketamine is a N- methyl-D-aspartate receptor (NMDA) antagonist and its underlying mechanism of is proposed to be in its ability to increase synaptic plasticity and this is ultimately believed to improve mood. On a molecular level, the antidepressant effects have been observed to be dependent on the activation of tropomyosin receptor kinase B (TrkB) signalling pathway. However, the antidepressant mechanism of ketamine remains still poorly understood as no new NMDA-antagonist or other rapid-acting antidepressants have been successfully developed for clinical use despite many years of effort. Therefore, some have proposed that the missing pieces of understanding its antidepressant effects might be linked to ketamine’s ability to modify sleep patterns and circadian-related molecules. Ketamine has especially been demonstrated to increase slow-wave activity during the following night of treatment and these changes have been shown to predict the clinical outcome in patients with major depressive disorder (MDD). Slow-wave activity is a low-frequency and high-amplitude wave seen in electroencephalography, which is highly expressed during the deepest stage of sleep, and this has been prominently found to be reduced in MDD patients. Even more intriguing, there are indications that ketamine might increase slow-wave activity also immediately after its administration. During this time, TrkB signalling is observed to became active. Following these molecular findings, we sought to investigate the link between the TrkB signalling pathway and two prominent processes occurring during slow-wave sleep. During slow-wave sleep processes such as (1) reduction of brain’s energy expenditure and (2) the activation of glymphatic system is known to occur. The glymphatic system is as lymphatic-perivascular network, which is responsible for clearing the brain from the metabolic waste. Thus, in this study, our objective was to investigate whether by causing an acute decline in adenosine-triphosphate (ATP) production or by stimulating the glymphatic network, we could activate the same plasticity-related pathways as ketamine is capable of activating in mice prefrontal cortex. The results of this study suggest that acute metabolic reduction can trigger pathways regarding synaptic plasticity. The metabolic inhibitor, 2-deoxy-D-glucose and mercaptoacetate (2DG+MA), was found to phosphorylate the TrkB receptor and its downstream signalling molecules GSK3β and p70S6K, while MAPK was dephosphorylated. These results correlate with the previous findings of ketamine’s effect after its administration. We also found a plasticity-related marker, MAP2, to be heavily phosphorylated by 2DG+MA, indicating 2DG+MA having a surprising role on neuroplasticity. These results are promising indication of understanding the rapid effects of ketamine and might even give important insight to developing novel antidepressants. However, these findings are only preliminary, and more research is needed to directly link antidepressant effects and energy metabolic inhibition together, as our study did not directly investigate antidepressants and depression-like behaviour in mice.
  • Snellman, Nana (2023)
    Chlamydia pneumoniae is an intracellular Gram-negative bacterium, that can cause respiratory infections. Infections are typically mild or asymptomatic, but it can also lead to more severe infections, for example, pneumonia. Severe infections might need antibiotic treatment. When the bacteria are exposed to stressful conditions, they can change to a chronic, persistent form. Amoxicillin and penicillin are known to transform bacterium into persistent forms. Antibiotics are not effective for persistent infection very often. Amoxicillin is the recommended treatment for pneumonia in Finland and worldwide, which is problematic from the perspective of C. pneumoniae. That is why there is a need for effective treatment for persistent C. pneumoniae infection. Probiotics and their by-products short chain fatty acids (SCFAs) are known to have beneficial effects on human health. Based on the current knowledge, SCFAs and other probiotic by-products are known to inhibit pathogen bacterial growth. Thus, SCFAs could have a potential effect on the treatment of C. pneumoniae infection. The aim of this work is to study the impact of SCFAs, acetate, propionate, and butyrate on C. pneumoniae infection and its antibiotic susceptibility. To study the impact of acetate, propionate and butyrate on C. pneumoniae infection and its antibiotic susceptibility, three different infection models were used: productive C. pneumoniae infection model with A549 cells, amoxicillin-induced persistent infection model with A549 cells, and persistent infection model with THP1 cells. Bacterial growth was followed with immunofluorescence and the number of the bacterial genome was studied with quantitative polymerase chain reaction (qPCR). The studied SCFAs did not have a significant impact on productive C. pneumoniae infection. With amoxicillin- induced persistent infection, the results were varying. For example, sodium acetate, and propionate showed some increase in bacterial growth on the first infection, but with sodium butyrate, there were not any impact. The only SCFA that decreased the bacterial growth in the persistent infection model with THP1 cells was sodium butyrate (200 μM). The same treatment also decreased the number of bacterial genomes with qPCR in the same infection model. In addition, the same condition increased the antibiotic susceptibility of persistent C. pneumoniae to azithromycin in THP1 cells. In conclusion, the studied SCFAs seemed to have more impact on C. pneumoniae infection with human immune cells compared to human lung epithelial cells. Based on this study, sodium butyrate could have positive impacts against persistent C. pneumoniae infection. Nevertheless, further studies of the impact of sodium butyrate on persistent C. pneumoniae infection are needed.
  • Savolainen, Heikki (2018)
    Tablet manufacturing requires both high-quality equipment and powder blend with high flowability and compactability and low segregation tendency. The process is complex and tablet formation process still remains not fully understood. Adequate powder flow is a necessity for the pharmaceutical manufacturing process, i.e., powder flowability and flow properties play a great role when designing manufacturing processes for solid dosage forms. As such, the powder characteristics need to be investigated. However, one property is seldom enough to predict the flowability of a powder in specific processes and different test methods need to be used to fully understand the tableting performance of a particular powder. It is crucial to know how the assessed properties reflect the manufacturing conditions. The need for test batches and the use of empirical testing still exists despite the numerous powder characterization tests available. The main aim of the study was to understand the influence of material properties, flow properties and segregation tendencies on both the processability of a formulation during tablet compression and the critical quality attributes, such as mass, tensile strength and dose uniformity of the final drug product. Additionally, testing of an in-line NIR method to observe the homogeneity of the powder inside the force feeder right before the compression step and transmission Raman as an at-line method for tablet content were also evaluated. A number of powder characterization tests were employed in order to fully understand the impact of the formulation on the process performance. Three formulations with different particle size of the active substance and mannitol were used throughout the study. Both the sifting segregation and fluidization segregation tests’ results predicted the formulations’ tabletability particularly well. Fluidization segregation test predicted the changing composition of the formulation throughout tableting whereas sifting segregation results showed the constantly fluctuating API concentration in the manufactured tablets. Moreover, the Raman results confirmed the tablets of variable content despite the offset caused by the different particle size of the raw materials used. The functionality of the NIR in the force feeder was tested successfully. The residence time distribution could be determined at a sufficient level to point out tablets of a bad quality from the batch on grounds of the NIR data. Results from the powder flow property tests were rather conflicting. Angle of repose, Carr’s index and volume flow rate gave the best characterizing results, whereas the mass flow rate, shear test with higher normal stress in pre-shear gave the worst results, considering the experienced flow character of the formulations. As stated above, different flow property tests may give conflicting result, and hence, it is crucial to know which results are the most relevant ones. Furthermore, the right settings for the test should be known to gain applicable results, best exemplified by the shear cell test.
  • Eriksson, Veronica (2020)
    Migraine was ranked as the second largest cause of disability in 2016 in the Global Burden of Disease (GBD) study. People with migraine have a greater disability and a lower health-related quality of life than those of the general population. Many migraine patients experience functional and emotional impairment due to their disease. Migraine can limit their daily activities and impact their private, professional and social life. Migraine affects the patient also in between the attacks and can impact their education, career and their family and loved ones. Comorbid diseases and failed treatment lines add to the burden of migraine. Furthermore, migraine also imposes an economic burden. Stigma is described as the hidden burden of disease. Chronic migraine patients have been found to have higher stigma than episodic migraine patients. Even though migraine is one of the most common disabling headache disorders, it is still both under-recognised, under-diagnosed and under-treated. The objectives of this study were to determine the extent of the burden and the stigma of migraine in adult Finnish migraine patients. This study aimed to produce comprehensive and current information about migraine and its severity in Finland, highlighting the burden it poses on the migraine patients as well as on society. Migraine is most prevalent among the working aged population, which increases the societal burden of the disease. This study was conducted as a cross-sectional electronic survey amongst adult Finnish migraine patients. The participants were contacted through the Finnish Migraine Patient Advocacy Group. The questionnaire consisted of the already existing and validated Migraine Disability Assessment (MIDAS) Questionnaire and of measures developed by the author. The final data consisted of 608 responses. Of all respondents with 8 or more headache days a month, over 90% were categorised in the severe disability group (MIDAS grade IV), thus having similar disability to those with 15 or more headache days a month (i.e. respondents with probable chronic migraine). The proportion of respondents with severe disability (MIDAS grade IV) was greater in the present study (65.0%) than in a study conducted in Finland in 2000 (47%), indicating that migraine disability in Finland might have become more severe during the past two decades. The mean level of headache pain in the present study was 6.2 (on a scale of 0-10) and pain was the aspect that most respondents viewed as the worst aspect of migraine. This highlights the importance of proper pain management in migraine care. Many of the respondents were also at risk for medication overuse, which highlights the importance of monitoring medication use and informing the patients about possible risks. Stress was reported as the most common migraine trigger, and reducing stress at the workplace was also reported as the most important way of how migraine could better be managed at the workplace. Almost half (44.4%) of all respondents felt stigmatised due to their migraine. Reasons for this stigma and suggested solutions on how to reduce/manage the stigma were quite similar. The ignorance of others was the most reported reason for their migraine stigma, and increasing awareness and correct information about migraine was the most reported way of reducing the stigma. Many of the respondents had faced, due to their migraine, belittlement at work, from family and friend and from healthcare professionals. Facing belittlement from healthcare professionals was reported to have happened often by 11.5% and sometimes by 34.7% of all respondents. Of all respondents, 55.6% worried often and 29.8% worried sometimes about the onset of the next migraine attack. The majority of the respondents had severe disability based on their MIDAS grades. Many other aspect of the burden were reported as well, inculding stigma, reported by almost half of the respondents. Further and future studies need to be conducted to get an even better understanding of the burden and stigma of migraine experienced by adult Finnish migraine patients. This includes further and more intricate quantitative and qualitative analyses of the data from this study, as´well as studies with new perspectives based on the results found in this study.
  • Silfvast, Saga (2016)
    Heart failure is a major public health problem and a leading cause of mortality worldwide. The most common cause of heart failure is myocardial infarction. Following a myocardial infarction, a large number of cardiomyocytes die and cardiac muscle is replaced by fibrotic scar tissue. Since the adult heart has inadequate endogenous regenerative capacity, loss of muscle tissue often causes a progressive decrease in cardiac function eventually leading to heart failure. At the moment heart transplantation is the only curative treatment for heart failure, but the low number of donor hearts is limiting the use of this treatment option. As current drugs only slow down the progression of the disease, there is a great need for new regenerative treatments. Direct cardiac reprogramming is a new approach for generating cardiomyocytes for cardiac regeneration. Unlike pluripotent stem cell-based strategies, direct reprogramming enables conversion of a terminally differentiated cell type directly into another cell type without first producing a pluripotent intermediate. Due to their abundancy and role in the repair of myocardial injury, fibroblasts represent an attractive starting cell type for direct cardiac reprogramming. Fibroblasts have been directly reprogrammed to induced cardiomyocytes (iCMs) by overexpression of key cardiac transcription factors, microRNAs (miRNA) or by modulating specific signal transduction pathways with small-molecule compounds. Despite successful reports of direct reprogramming both in vitro and in vivo, the efficiency of direct reprogramming remains, however, too low for potential clinical applications. The aim of this M.Sc. thesis work was to establish direct reprogramming of mouse embryonic fibroblasts (MEFs) to iCMs by viral overexpression of cardiac transcription factors Hand2 (H), Nkx2.5 (N) Gata4 (G), Mef2c (M) and Tbx5 (T) and a small-molecule compound screening platform for identifying small-molecule compounds that could enhance the reprogramming efficiency and potentially replace cardiac transcription factors in direct cardiac reprogramming. In accordance with previous publications MEFs were successfully directly reprogrammed to iCMs using both HGMT and HNGMT cardiac transcription factor combinations. The screening platform was tested using the TGF-β inhibitor SB431542, which has recently been reported to increase the cardiac reprogramming efficiency. In line with previous publications, the reprogramming efficiency was significantly increased by treatment with SB431542. Initial tests with other small-molecule compounds did not have a positive effect on the reprogramming efficiency. The results of this M.Sc. thesis work verify previous publications and demonstrate a method for in vitro small-molecule compound screening, which can be used to identify compounds that increase the reprogramming efficiency in direct cardiac reprogramming. However, the results shown here are only preliminary and more replicates are needed in order to confirm the current results. Nonetheless, the results of this thesis work set a foundation for finding small-molecule compounds that in the future might be used to target direct cardiac reprogramming as a regenerative therapy for myocardial infarction and heart failure.
  • Kahma, Helinä (2014)
    Active transport processes in the basolateral (sinusoidal) membrane of hepatocytes have an important role in the hepatic clearance and overall disposition for several types of drugs. Organic anion transporting polypeptides (OATPs) expressed in the sinusoidal membrane have been shown to mediate the sodium-independent hepatic uptake of broad range of drugs and they have been associated with clinically relevant drug-drug interactions (DDIs) and genetic polymorphisms. The literature review focuses on sinusoidal OATP transporters and on the pharmacokinetic effects of OATP-mediated hepatic uptake. In addition, current methods to investigate the interactions between drugs and transporters are discussed, with the emphasis on methods applicable to study uptake transporters. The aim of the experimental part of the master's thesis was to determine if two clinically used drugs, entacapone and fluvastatin, are actively transported from blood into rat and human hepatocytes, and to assess the role of OATP transporters in the hepatic uptake of the drugs in comparison with known OATP substrates, estrone 3-sulfate (E3S) and taurocholic acid and broad OATP inhibitor rifamycin SV. The uptake kinetics of compounds of interest were determined in freshly isolated and cryopreserved rat hepatocytes and in cryopreserved human hepatocytes using the oil-spin method. Uptake clearances (CLuptake) via active uptake (CLactive) and passive diffusion (Pdiff) were calculated from the initial uptake data over a 1 - 200 µM and 1 - 50 µM concentration range for entacapone and fluvastatin, respectively. The half-maximal inhibitor concentration (IC50) of E3S uptake transport was determined for entacapone in a competitive uptake experiment over a 10 - 400 µM concentration range. Fluvastatin uptake showed active saturable transport kinetics in rat hepatocytes with a Km value of 6 µM, whereas entacapone uptake in rat hepatocytes was somewhat linear and did not inhibit E3S uptake at clinically significant concentrations, with an IC50 value of 240 µM. Significantly lower hepatic uptake of taurocholate and entacapone was observed between rat and human hepatocytes, indicating species differences in hepatic uptake processes, although cryopreservation may have had an effect on the noticed difference. The results suggest that murine Oatp transporters do not have a significant contribution to hepatic uptake of entacapone. However, this should be confirmed with future studies with more repetitions and a reliable quantification method.
  • Kouri, Riikka (2011)
    The p53-family consists of three transcription factors, p53, p73 and p63. The family members have similar but also individual functions connected to cell cycle regulation, development and tumorigenesis. p53 and p73 act mainly as tumor suppressors. During DNA damage caused by anticancer drugs or irradiation, p53 and p73 levels are upregulated in cancer cells leading to apoptosis and cell cycle arrest. p53 is mutated in almost 50 per cent of the cancers, causing the cancer cells unable to undergo cell death. Instead, p73 is rarely mutated in cancer cells and because of that could be more viable target for anticancer therapy. The network surrounding the regulation of p73 is extensive and has several potential targets for cancer therapy. One of the most studied is Itch ligase, the negative regulator of p73 levels. Gene therapy directed towards knockdown of Itch ligase is a potential approach but in need for more in vivo proof. p73 has two isoforms, transactivating TA-forms and dominant-negative ΔN-forms. The specific regulation of these isoforms could also offer a possible way for more effective cancer treatment. The literature work includes information of structures, isoforms, functions and possible therapeutic targets of p73. Also the main therapeutic approaches to date are introduced. The experimental part is based on transfection and cytotoxicity studies done e.g. in pancreatic cancer cells (Mia PaCa-2, PANC1, BxPc-3 and HPAC). The aim of the experimental work was to optimize the conditions for effective transfection with DAB16 dendrimer nanoparticles and to measure the cytotoxicity of plain dendrimers and DAB16-pDNA complexes. Also the protein levels of p73 and Itch ligase were measured by Western blotting. The work was done as a part of a bigger project, which was aiming to down regulate Itch ligase (negative regulator of p73) by siRNA/shRNA. Tranfection results were promising, showing good transfection efficacy with DAB16 N/P30 in pancreatic cancer cells (except in BxPc-3). Pancreatic cancer cells showed recovery in 3 days after they were exposed to plain dendrimer solution or to DAB16-pDNA. Measurement of protein levels by Western blotting was not optimal and the proposals for the improvement regarding e.g. the gels and the extracted protein amounts have been done.