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(2012)Solid materials can exist in two major forms: in crystalline or amorphous form. Amorphous form is defined as no long term order existing in solid structure in molecular scale. Amorphous materials have different physicochemical properties compared crystalline forms of same substance. Amorphous materials doesn't have sharp melting point as crystalline materials. When heated above so called glass transition temperature amorphous materials become rubbery (plasticization) and when cooled below they become glassy (hard and brittle). Amorphous forms can also have different dissolution properties which makes them useful in formulation of poorly soluble drugs. Amorphous forms are less stable compared to crystalline form. That's due amount of free energy stored in it's structures. Amorphous materials can be manufactured in many ways including quench cooling, hot-melt-extrusion, spray drying and lyophilisation (freeze drying). In experimental section effect of grinding method in properties of amorphous indomethacin was studied. Amorphous indomethacin was prepared by quenching of melt in liquid nitrogen. Properties of amorphous indomethacin was studied by x-ray powder diffraction and differential scanning calorimetry. Measurements were performed in different time stamps varied form 0 to 92 days. Measured properties were crystalline content, glass transition temperature, change in heath capacity, heat of crystallization, heat of melting and melting points of crystallized forms. Calorimetry data was recorded only from totally amorphous samples. It can be seen in results that different patches are not comparable statistically but when comparing room temperature ground and liquid nitrogen ground samples to each other differences can be found in every set. Difference is observed in initial time of crystallization (time when crystallinity can be measured first time) and in thermodynamical properties such as change in heat capacity, glass transition temperature and heat of melting. Solid dispersions of indomethacin and xylitol were prepared in 3 different compositions (5%, 10% and 20% xylitol in indomethacin). XRPD and DSC data were measured at different time stamps (aged 1 to 63 days). 5% and 10% dispersions found to be stabile and being amorphous in all time stamps. 20% dispersion was already partly crystallized at 63 days (especially liquid nitrogen ground sample).
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(2014)One way to improve the solubility of a poorly-water-soluble drug is to make amorphous solid dispersion of it with one or several carrier polymers. However, the amorphous solid dispersions are often unstable. Stability and amorphisation of drug substance depend on e.g. the miscibility of the components in dispersion. Moreover, in the early stage of drug development there is available only limited amount of active substance and time to the analyses. In this study, the primary goal was to develop a method combining the preparing (solvent method) and the analyzing (MTDSC, modulated temperature differential scanning calorimetry) methods. In the method developing part, the possible effect of analyzing parameters of MTDSC to the results was also tested. Amorphous solid dispersions were prepared and analyzed with the invented method. The dispersions were made of poorly-watersoluble itraconazole with hydroxypropylmethylcellulose acetate succinate (HPMC-AS) and/or polyvinylpyrrolidone (PVP K30). X-ray powder diffraction (XRPD) and polarized light microscopy (PLM) were also used to make the interpretation of results easier and more reliable. By analyzing the prepared dispersions the differences in the miscibilities of the used polymers with itraconazole were examined and it was also studied how the miscibility affected to the amorphicity of the prepared dispersion. As a secondary goal, it was tested if combining the two polymers would improve the miscibility and amorphicity of the prepared dispersion. In many cases, with the developed method it was possible to make mixed and amorphous solid dispersion with 10-20 % itraconazole concentration. Used small amount of drug was roughly enough to the detection limit of the used analyzing techniques. The analyzing parameters of MTDSC were not seen to affect to the results in this study which makes the use of this method easier. The results of used analyses were in some part contradictory and that is why it is recommended to use several analyzing techniques or methods that combine different kinds of techniques. In the study, it was seen that in the most part of the prepared dispersions there was more HPMC-AS than PVP K30. This was speculated to be caused by the ionic bonds between the basic itraconazole molecules and acidic succinyl groups in HPMC-ASs and also because of more hygroscopic nature of PVP K30 which increases mobility which in turn increases probability of collision of itraconazole molecules. The use of two polymers in the same time was useful especially in the case of 90/10 HPMC-AS/PVP K30 polymer ratio. This was speculated to be caused by different vaporization rates of the used solvents (DCM and methanol) and too slow evaporation phase. To explain and examine this observation more thoroughly, nuclear magnetic resonance (NMR) -measurements were done. When analyzing the prepared dispersions and itraconazole alone, it was observed that with used amorphisation method (solvent method) itraconazole was in a form that differs from the original polymorph. This form of itraconazole was probably some kind of liquid crystal and was examined further by heating the sample and analyzing it by XRPD. Although there are some other studies to support this hypothesis, this interpretation needs some confirmatory analyses with other methods: with high temperature SAXS (small angle X-ray scattering) and NMR.
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(2015)Today, many of the new drugs are poorly soluble in water, which can be a problem in the drug development. Solid dispersion is a formulation technique, which improves the dissolution rate of the drug. However solid dispersions, where the drug is in amorphous form, are often unstable. Because of that, solid dispersions, where the drug is in crystalline form, have been developed. Drug crystallization and factors affecting to the crystallization, such as amount of the polymer, are important to examine to be able to develop better drug products. Different kinds of mathematical models, which describe the kinetics of crystallization, has been developed to help to understand the crystallization event more comprehensively. In this study, the crystallization of the amorphous drug, in the absence of polymer and with a low polymer concentration, was investigated. The crystallization was also examined using a mathematical model designed to determine the kinetics of crystallization in order to find out does it work in this case. A model drug was felodipine and polymers used in this study were HPMCAS-LF and PVP K30. The concentration of polymers in the solid dispersions was 10% and 20%. It was found that a small amount of polymer has a very significant effect on crystallization rate of felodipine. Mathematically defined crystallization rate constant k increased by 13 times, when the amount of PVP was decreased 20 % to 10 %. The polymer concentration also had an effect on nucleation time which is the time before crystallization occurs. For example in the solid dispersion, where PVP concentration was 10 %, the nucleation time was five times slower and 20 % PVP consentration ten times slower than felodipine alone. The work also showed that HPMCAS stabilizes the amorphous state of felodipine better than PVP at 40 ° C / 75% RH conditions. This was observed in both MTDSC-measurements and the polarizing light microscopy. The difference between polymers was thought to be due to weakening of the interactions between PVP and felodipine by the influence of water in humid conditions. However, the different formulations had no significant effect on dissolution characteristics of felodipine. There is a possibility that felodipine crystallizes at the beginning of dissolution. It should be noted that mathematical method tested was not able to model crystallization kinetics properly in this study. So care should be given, when using a mathematical model in the product development.
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(2020)Amyotrophic lateral sclerosis (ALS) is a rare fatal neurodegenerative disease in which both the upper and lower motor neurons degenerate. Pathological features of the disease include misfolded proteins and accumulations in the central nervous system. The molecular mechanisms of the disease include neuroinflammation, glutamate induced excitotoxicity, and endoplasmic reticulum stress (ER-stress). Numerous genetic defects have been identified in the background of ALS, the most common mutations are in the C9ORF72, SOD1, TDP43 and FUS genes. For each gene mutation, it is important to develop a reliable animal model of ALS for studying pathology and testing new therapies. The most common and most recently found gene mutation, the C9ORF72 repeat expansion mutation, does not yet have an established animal disesase model. The molecular mechanisms of the disease include neuroinflammation, glutamate induced excitotoxicity, and endoplasmic reticulum stress (ER- stress). There is no drug treatment to cure or slow ALS, so the need for new drug therapies that affect the course of the disease is significant. Cerebral dopamine neurotrophic factor (CDNF) protects and restores dopamine neurons and controls ER-stress in preclinical models of Parkinson’s disease. CDNF has also been shown to improve motor coordination as well as protect spinal cord neurons from cell destruction in ALS genetic SOD1- G93A mouse and TDP-43M337 animal models. The purpose of this master's thesis study was to characterize the changes related to neurodegeneration and neuroinflammation in the new C9ORF72-500 disease model and study ER stress of the SOD1-93A disease model and the effect of CDNF on ER stress in SOD1-model and on inflammation in C9-model. In the first sub-study, brain sections from C9ORF72 transgenic and wild-type mice at different time points were subjected to six different immunohistological stainings. The results were compared at each time point (30, 70 and 170) between the wild type and the transgenic group. In another sub-study, spinal cord sections from CDNF snd vehicle treated SOD1- G93A mice were subjected to immunofluorescence staining, after which the intensity of their ER stress marker, GRP78, was analyzed using a confocal microscope. GFAP stained brain sections from CDNF and vehicle treated C9ORF72 mice were analyzed using microscope and imaging analyses. The results of the first sub-study showed neuroinflammation at 24 weeks timepoint in the transgenic group compared to wild-type mice. Pathological features of C9-ALS, various protein accumulations, were observed only in the transgenic group, mainly at 24 weeks. No neuronal loss was observed in this study. The obtained results support the previously published research results and support the reliability of the studied disease model. In the second sub-study ER stress levels were higher in SOD1-mice compared to wild-type mice. Single intracerebroventrical CDNF injection reduced ER stress in SOD1-G93A transgenic mice almost to the same level as ER stress in wild-type mice. CDNF treatment also showed a tendency for reducing inflammation in hippocampus and motor cortex of C9ORF72 mice. The results confirm the pathological role of ER stress in ALS and show that CDNF reduces ER stress when administered as early in the disease as possible, when neuronal damage begins to occur but does not yet lead to neuronal destruction. CDNF appears to be a promising drug candidate for the treatment of ALS and should therefore be further investigated.
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(2024)General anaesthetics are pharmaceutical agents used to induce general anaesthesia, a reversible state of unconsciousness. Caenorhabditis elegans, a nematode species, has been successfully used as a model organism in the study of gaseous anaesthetics due to its amenability to genetic modification, fully mapped nervous system connectome and high evolutionary conservation. However, C. elegans is less well characterised as a model organism in the study of non-gaseous anaesthetics. The primary aim of the study was to study the potential of ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, and urethane, a nonselective modulator of various neurotransmitter-gated ion channels, to immobilise C. elegans without activating antioxidant response signalling by transcription factor SKN-1, the nematode orthologue of the mammalian Nrf/CNC proteins. The transparent body of C. elegans enables microscopic imaging of cellular processes, but high-quality imaging requires the immobilisation of the worm. A commonly used chemical immobilising agent, sodium azide, causes SKN-1 activation in C. elegans, which may limit the use of sodium azide in studies on SKN-1 promoters. A secondary aim of this study was to study the impact of ketamine, an activator of mTOR (mechanistic target of rapamycin) signalling, on the lifespan of C. elegans. The lifespan of C. elegans has been found to increase with inhibition of the mTOR homologue pathway in previous studies. Ketamine and urethane were administered to wild-type C. elegans in aqueous media in 96-well plates. Behavioural endpoints of immobility and uncoordination were assessed manually via microscopic observation and video recordings. SKN-1 activation was studied by measuring drug-induced fluorescence in mutant strain CL2166, which carries a green fluorescent protein reporter of SKN-1 downstream target GST-4 (glutathione-S-transferase). A lifespan assay was performed with sterile C. elegans strain SS104 by incorporating ketamine in the worm maintenance agar. In this study, urethane did not appear to be a potent immobilisation agent. Ketamine was found to cause reversible weak immobilisation at a similar concentration at which sodium azide fully paralyses wild-type C. elegans. At lower doses ketamine caused uncoordinated locomotion. Short-term exposure to an immobility-inducing dose of ketamine was not found to significantly activate SKN-1. In the lifespan assay, ketamine unexpectedly appeared to significantly increase the nematode lifespan compared to control treatment.
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(2023)Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by the death of nigrostriatal dopaminergic neurons and formation of intraneuronal protein aggregates called Lewy bodies and Lewy neurites. These inclusions consist of a protein called α-synuclein (aSyn) but also of other proteins, lipids and cell organelles. Progressive cell death leads to nonmotor and motor symptoms. Current therapies for PD are symptomatic and do not modify the disease progression. Therefore, there is a need for the development of therapies attenuating the neurodegeneration. The pre-formed fibrils (PFF) model enables studying of aSyn aggregation and mechanisms behind inclusion formation. The PFF model is based on the exogenous aSyn fibrils’ tendency to result in formation of Lewy body -like inclusions when added in cell culture or in animals. Primary neuronal cultures of mice and rats have typically been used to model aSyn aggregation in vitro with the PFF model. Primary neuronal cultures provide practicality and are able to depict relevant features of dopaminergic neurons. To gain insight about the composition of E13.5 primary embryonic mouse midbrain culture and to enable adaptation of an existing protocol to study other cell types, this study identified and quantified several relevant cellular phenotypes in the micro island culture. The cells were fixed on day in vitro (DIV) 8 or DIV 22 and analysis was conducted using fluorescent immunocytochemistry combined with automated image analysis software, CellProfiler. On DIV 8, tyrosine hydroxylase -positive dopaminergic neurons represented 5 % of the total cells in the culture. Neuronal nuclear antigen -positive neurons resulted representing 30 % of the total cells. Gabaergic neurons were identified to be abundant in the culture and certain dopaminergic neurons were identified as immunoreactive for GABA. Choline acetyltransferase -positive cholinergic neurons were also identified to be present in the culture. The number of oligodendrocyte precursors (OPCs) was observed to be significantly smaller than the number of dopaminergic neurons. OPCs represented around 1 % of the culture on DIV 8. Glutaminergic neurons, parvalbumin-positive interneurons, microglia or astrocytes were not identified in the culture on DIV 8. The number of astrocytes was observed to increase as the incubation time was prolonged to DIV 22. Overall these findings provide valuable insights of the composition of cell phenotypes in E13.5 mouse midbrain culture. The results also provide additional validation for suitability of the original protocol to robustly produce midbrain dopaminergic cultures with minimal number of glial cells. Understanding more about the relevance and interplay of different cell phenotypes in PD pathophysiology can provide valuable insight for the development of potential therapeutic strategies.
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(2012)This thesis is constructed as a part of a larger research project aiming to increase understanding of polyketone reductases (PKR) and develop applications from them. PKRs are enzymes in biosynthetic pathways leading to several aromatic secondary metabolites in plants. The previous work in the research group has led to establishment of several callus cultures from plants belonging to the genus Rubus in the family Rosaceae. The aim in the experimental part of this thesis is the identification and semi-quantitation of raspberry ketone (RK) and related aromatics in the cell suspension cultures initiated from the previously established callus cultures. RK is biosynthetically produced by reduction of p-hydroxybenzalacetone (p-OH-BA) by benzalacetone reductase (BAR). As a part of the experimental work, p-OH-BA has to be chemically synthetized and analysed. Special emphasis is placed to experiment, develop and validate an extraction method for phenolic compounds using ASE 200 working station. In the review part of this thesis, the basic procedures of chemical analysis are described, optimization and validation of analytical methods are discussed, and lastly studies related to raspberry ketone (RK) are summarized. The detection limit is 0.73 µg/ml for RK with the established UPLC-UV method, and the quantitation limit (QL) is 2.22 µg/ml. At the QL, the standard deviation of the extraction method is 8.9 % and the results are 6.4 % higher than expected. At the high end of the standard curve the extraction results are 18.7 % higher than expected. Some changes are proposed to optimize the method. Analysis of the cell line extracts with the established UPLC-UV method did not readily reveal any of the studied compounds. Although the interpretation of the results of the MS experiment is still underway, RK was detected from the arctic bramble cell line Ra15. Also, a possible derivative of zingerone was detected from cloudberry cell line extract even without the corresponding standard compound. This shows the power of the MS in metabolite profiling, and gives a course for future studies.
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(2023)Diseases of the posterior eye segment, such as age-related macular degeneration (AMD), diabetic retinopathy, diabetic macular edema and glaucoma are the leading cause of blindness worldwide. Current therapy to treat these vision-threatening diseases relies on intravitreal injections to maintain a desired therapeutic drug concentration in the back of the eye. Frequent intravitreal injections are uncomfortable with poor patient compliance and causes major burden to the healthcare systems as well as to the patients. Small molecule drugs have shorter half-life in the vitreous and are eliminated rapidly. This requires frequent intravitreal dosing intervals that are not feasible in the clinical settings. Also, intravitreally injected small molecule drugs are often poorly and non-specifically distributed to the ocular tissues causing adverse effects. To address these issues, controlled and sustained drug delivery systems in the form of drug conjugates are desirable. Conjugating small molecule drugs with enzymatically cleavable peptide linkers increases the residence time in the vitreous. The peptide linker gets cleaved by vitreal enzyme and the released drug reaches the target in retina and choroid. Aim of this thesis was to screen a library of 25 peptide linkers for cleavage in the presence of porcine vitreal enzymes. The peptide linkers were chemically synthesized and the in vitro stability of the peptide linkers were studied in freshly isolated porcine vitreous. Ten time point samples were collected over a period of 45 days and the peptide cleavage in porcine vitreous was assessed by LC-MS method. A TQ-S liquid chromatography-mass spectrometer was used to study the linker cleavage. LC-MS method development for the peptide library was carried out using IntelliStart wizard function. Out of the 25 peptide linker in the library, stability of eight linkers were not included in the LC-MS analysis as a mass method could not be developed. Out of 17 peptide linkers studied, 14 were categorized as fast cleaving linkers (>90% of the linker cleaved in porcine vitreous after 5 h). Three linker peptides; P4, P5 and P25 were categorized as slow cleaving linkers. Conjugating slow cleaving peptide linkers to small molecule drugs will increase the half-life and enhance the duration of drug action upon intravitreal injection. In this study, linkers that are hydrolyzed by specific enzymes present in vitreous or ocular tissues are exploited to investigate their potential for delivering small molecule drugs.
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(2020)Monoclonal antibodies (mAbs) are widely used in the treatment of several diseases such as cancer and autoimmune diseases. Due to their high prices and growing consumption, therapeutic mAbs have become potential targets of falsification. This generates a demand for quick and efficient analytical procedures for identifying and characterizing mAbs in a case of suspected falsification. The structure of therapeutic mAbs consists of human or murine IgG framework, where unique complementarity determining regions (CDRs) are engineered with different recombinant techniques. Given the complex nature of the mAbs, they must be identified using multiple complementary analytical methods. Ten full-sized therapeutic mAbs, Fab-fragment abciximab and CTLA4-Fc-fusion protein belatacept were studied in order to find analytical methods for efficient characterization and identification. All studied antibodies were characterized by their charge and molecular weight by isoelectric focusing (IEF) in polyacrylamide gels, native and reduced SDS-PAGE, and size exclusion chromatography (SEC). Six mAbs, abciximab and belatacept were digested with trypsin, and the cleaved peptides were further analysed by RPLC-MS. In addition, quantification methods including SEC peak area measurements and Bradford protein assay were performed for all antibodies. As expected, SDS-PAGE of non-reduced and reduced mAbs gave little distinction between the mAbs. Both methods were however shown to be useful in the identification of the mAb nature, as they confirmed the presence of heavy chains, light chains, and disulfide bonds. IEF showed potential in mAb identification, as clear, partly distinguished patterns of charge variants were obtained. However, some improvements to the pH gradient are needed to enable better separation and pI estimation of basic variants. Determination of molecular size with SEC was found to be difficult, as there seemed to be no consistency between the calculated molecular weights based on measured elution times, and the theoretical molecular weights. Nevertheless, SEC brings added value in mAb quantification and detection of protein aggregation and fragmentation. Finally, RPLC-MS analysis of tryptic peptides resulted in mAb identification, with the measured sequence coverage of 87-97 %. Identification process may be enhanced by focusing on the known CDR-peptides prior the constant frame peptides. Given the structural similarity of therapeutic mAbs, identification of an unknown mAb requires combination of multiple analytical methods. If available, the use of reference mAb product obtained from a reliable source is recommended, as the identification may be based on comparative analyses using simpler analytical steps, e.g. IEF, SDS-PAGE and SEC. If no reference product is available, identification of the mAb requires peptide mapping and determination of the CRD sequences by RPLC-MS analysis. Further research is needed to find a suitable set of analytical methods for identification of all therapeutic mAbs.
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(2020)The solubility of a poorly water-soluble drug can be improved by converting the crystalline drug into an amorphous form. However, the amorphous form is metastable due to the higher energy state and recrystallization may occur during storage and dissolution. The amorphous form can be stabilized by forming an amorphous solid dispersion (ASD), where the drug molecules are dispersed to the solid medium, e.g. hydrophilic polymer. One preparation method for amorphous solid dispersions is spray drying, where a solution containing a drug and polymer is converted into small droplets in a drying chamber, in which the solvent evaporates in a hot gas stream and solid particles are formed. The aim of this study was to investigate whether an ASD of a poorly water-soluble drug can be prepared by spray drying using 20:80 (V/V) ethanol-water mixture as a solvent in a feed solution. Indomethacin (γ-polymorph) was used as a model drug and polyvinylpyrrolidone vinyl acetate (PVPVA) as a polymer. The aim was to find a suitable formulation where the drug is in the amorphous form after spray drying and remains in the amorphous form during storage. The ratios of the drug to polymer in the spray-dried formulations were 1:4, 1:6, 1:8, 1:10, 1:12 and 1:16. The study also examined whether a change in one process parameter, pump feed rate, affects the amorphous nature and stability of the resulting spray-dried solid dispersions. Two different pump feed rates, a higher 30% and a lower 15%, were used in the study. X-ray powder diffraction (XRPD) was used to characterize the solid-state of the spray-dried formulations. XRPD measurements were performed immediately after spray drying and on selected time points during storage. Formulations 1:10 at 30% feed rate, 1:12 at both feed rates and 1:16 at 30% feed rate were amorphous after spray drying. In 1:12 (30%) and 1:16 (30%) formulations indomethacin remained in amorphous form over the study periods (22 and 56 days, respectively). In other formulations, indomethacin was found to be in crystalline α-form immediately after spray drying or recrystallization to the α-form occurred during storage. The interaction between indomethacin and PVPVA was studied by surface plasmon resonance spectroscopy (SPR). The aim of the SPR measurements was to understand the interaction between these substances in the feed solution used in spray drying. PVPVA solutions of various concentrations (1%, 0.5%, 0.1% and 0.01%) were injected to the surface of the gold sensor coated with crystalline γ-indomethacin, and the changes in the SPR signal responses were monitored during the interaction. The same measurements were also performed on a pure gold sensor without indomethacin. An interaction between indomethacin and PVPVA can be observed, and based on the measurements, a polymer layer with a thickness of about 1 nm was formed on the surface of the indomethacin sensor regardless of the concentration of the polymer solution. Thus, even a small amount of polymer in solution is sufficient to cover the indomethacin crystals. This may also occur in the feed solution during spray drying, but further studies with SPR are still needed, especially with amorphous indomethacin. This study showed that an ASD of indomethacin and PVPVA can be successfully prepared by spray drying using an aqueous feed solution. Spray-dried 1:12 and 1:16 formulations at a higher pump feed rate were found to be stable enough for further studies. If the spray-dried material is further formulated into a pharmaceutical product, indomethacin must remain in amorphous form throughout the shelf-life of the product to maintain the improved solubility.
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(2020)Endothelial dysfunction is a common characteristic of several diseases including diabetes mellitus, coronary heart disease and stroke. Healthy endothelium ensures vascular homeostasis, regulation of blood flow and the exchange of oxygen and nutrients, as well as immune cell filtration to the surrounding tissues. In many cases, endothelial dysfunction results in ischemia in the surrounding tissues impairing cellular regeneration mechanisms, which can lead to tissue necrosis in the worst case. Therapeutic angiogenesis via stem cell transplantation aims to restore tissue blood flow and thus aid in tissue regeneration and restoration of a functioning tissue. Adipose derived stem/stromal cells (ASC) are a stem cell population with a multilineage differentiation ability. They have been shown to differentiate towards adipogenic, osteogenic, chondrogenic, myogenic and neurogenic lineages among others. Their easy obtainability from liposuction material and abundance in the adipose tissue makes them an especially practical and favorable cell option for stem cell research. In angiogenesis research, ASCs are commonly used in a co-culture with an endothelial cell (EC) type such as human umbilical vein endothelial cell. ASCs secrete extracellular vesicles (EV) that are small membrane bound vesicles with a diameter ranging from 40-1000 nm, and which have the ability to alter the behavior of target cells through their cargo. EV cargo consists of microRNAs, messenger-RNAs and proteins, and the EV cargo of ASCs has been shown to have proangiogenic effects. The aim of this work was to review what is currently known about ASC ability to promote angiogenesis through paracrine secretion and differentiation into endothelial cells or pericytes, interactions between ASCs and endothelial cells in the angiogenesis promoting process and the role of ASC extracellular vesicles in promoting angiogenesis. The methods for this work were database research of related articles using scientific databases and search engines, article categorization and reading, and finally manuscript production. It can be concluded from the current literature that a co-culture environment of ASCs and an endothelial cell type supports the formation of tube-like structures in vitro. Additional insulin like growth factor 1 in culture medium enhances the expression of angiogenesis-related growth factors in both cell types via PI3K/AKT signaling pathway. Further, the activation of platelet derived growth factor receptor β supports ASC ability to promote vascular network formation. On the contrary, the presence of ASC secreted activin A results in the inhibition of vascular network formation. ASCs can differentiate into endothelial cells particularly in three-dimensional culture conditions. In addition, fibroblast growth factor 2 and the activation of the AKT-pathway are crucial for endothelial differentiation. In addition, ASCs have the ability to differentiate into pericytes and assume a stabilizing role on the outside of the microvessels. Concerning ASC derived EVs and their cargo, miR-31, miR-125a and miR-126 have proangiogenic effects in vitro and in vivo. Proangiogenic miRNAs in ASC EV cargo are miR-181b-5p and the let7-family, out of which miR-181b-5p upregulates vascular endothelial growth factor and hypoxia-inducible factor 1α and let7-family influences tube formation ability of ECs. In vivo, ASC derived EVs support fat grafting, enhance wound healing both in healthy and diabetic environment, and provide cardioprotection. Therefore, ASC EVs show potential for therapeutic angiogenesis but currently there is a lack of clinical trials in EV research.
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(2022)Introduction: When people age, the composition of sleep changes and sleep becomes more sensitive to external disturbances, making insomnia also more common. Medication is not the first-line treatment option for insomnia. Benzodiazepines or benzodiazepine receptor agonists for the treatment of insomnia have been in the focus of past studies. The content of the dosing instructions for the supplied medicines has not been studied. The dosing instructions should provide clear instructions on how to dose the medicine prescribed to the patient. The aim of this study was to investigate the content and quality of dosing instructions prescribed for the treatment of insomnia for Finns aged ≥75 years in 2020 based on the prospective reimbursement register data by the Social Insurance Institution of Finland (Kela). Materials and methods: The reimbursed purchases of all medicines by persons aged ≥75 years from 1.1.2020 to 31.12.2020 were selected by ATC code from the medicines data according to the Insomnia: Current Care guidelines. The data was gathered from Kela’s dispensations reimbursed under the National Health Insurance scheme. The dataset consisted of 1,080,843 delivery lines, which were screened, and 328,285 lines were included in the analyses. Dosage instructions were reviewed according to the following predetermined five categories: frequency of use, dose, timing, warnings or remarks, and inappropriate instructions. In addition, 1000 dosing instructions were randomly derived to study the phrasing and appropriateness of the dosing instructions in more detail. Results: In 2020, an average of 3.8 reimbursed hypnotic drug deliveries were made per elderly person. Of the deliveries, 68% were for women. 52% of drug deliveries were partially made later and not by original prescription. In the hypnotic dataset, the three most administered drugs were zopiclone (41%), mirtazapine (34%) and zolpidem (12%). The dosage was prescribed in 98–99% of the dosage instructions. Dosing schedule was reported in 83% of dosing instructions and regularity of use was reported in 57% of them. Only 3-6% of the dosing instructions had comments or warnings. 1–2% of the dosing instructions were vague. The duration or regularity of use was clearly indicated in 5% of the dosing instructions. Only 0.1% of dosage instructions contained instructions for discontinuation or reduction. Discussion: The dose and timing of administration were well reported, but the frequency of use was reported in only about half of the dosing instructions. Only few dosing instructions contained remarks or warnings even though hypnotics are at risk for the elderly. Among the three most administered drugs for the treatment of insomnia were two benzodiazepine receptor agonists, zopiclone and zolpidem. However, they may not be suitable for the elderly according to Beers criteria and their use should be avoided. During 2020, an average of four drug deliveries were made per elderly person for the treatment of insomnia, which may indicate prolonged hypnotics use. In addition, more than half of the deliveries were partially made later and not by original prescription. Thus, several drug packages are prescribed for prescriptions, although the drug-based treatment of insomnia should only be short-lived. Conclusions: There are significant deficiencies in the contents and quality in the dosing instructions for drugs delivered to the elderly for insomnia. Minimum information on dose, timing and duration of use was not found in all dosing instructions in this study. Understandable dosing instructions and the reduction in the amount of medication in the prescription could have a further effect on reducing the long-term use of hypnotics, also increasing the safety of medicine use in the elderly.
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(2016)Nowadays, there is still lack of commercially produced drugs for children. Extemporaneous compounding is needed widespread. Oral powders, capsules and oral suspensions are the most typical extemporaneous dosage forms. In Finland, oral powders have traditionally been the most used. Major concern relating to the extemporaneous products is that they are not authorized. That means that their safety and effectiveness have not been established. Compounding oral powders is time consuming and their overall mass is much higher compared to capsules with same strength. That increases the amount of foreign matter in child patients, which is highly not recommended. The aim of this study was to examine, whether the extemporaneous sotalolhydrochloride capsules meet European Pharmacopoeia standards of content uniformity. Additionally, because the feeding tubes are widely used in neonatal patients, it was also reasoned to examine the content uniformity of capsules lead through the feeding tube. A significant part of this study was to develop an accurate and effective HPLC -method for analyzing sotalolhydrochloride, which, in the end, turned out well. With its seven minute driving time per sample, it is suitable even in routine analysis. Two of three capsule batches, as well as the oral powders, met the European Pharmacopoeia standards of content uniformity. Also, leading the capsule contents through the feeding tubes met the standards, but the amount of drug substance was significantly lower compared to capsules and oral powders. With lower overall mass and being quicker to prepare, capsules are recommendable option for traditional oral powders in extemporaneous children's medication. Still, according to this study, it is important to take into consideration the possibility of excessive variation in content uniformity. Thus, in the future, it is necessary to develop the quality control systems in hospital pharmacies.
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(2009)Anticholinergic medicines are commonly used to treat e.g. incontinence. These medicines have side effects, which may cause and also exacerbate e.g. dryness of the mouth, increased heart rate, and even cognitive impairment. Older people may be more at risk for these side effects as they may be experiencing similar symptoms as a natural effect of aging, and because they may be using several medicines causing these effects. Older people often have a high medicine burden and also a high disease burden. Measuring anticholinergic effects to change medicine regimens and to reduce the symptoms is difficult as there is no golden standard method. This thesis investigated the published methods available for estimating anticholinergic burden in the literature review part, and used one anticholinergic scoring system, the Anticholinergic Risk Scale, in a cross-sectional study to test the effects of anticholinergics on mortality in 1004 older institutionalised patients from Helsinki area public hospitals. Cross-tabulations and Kruskal-Wallis or Chi square methods were used to detect differences between variables such as nutritional status or certain diagnoses when the patients were stratified according to their anticholinergic use. Cox Proportional Hazard regression, the logrank test and Kaplan-Meier curve were used to investigate the effects of anticholinergics on 5-year all-cause mortality. An in vitro serum assay and seven anticholinergic scoring systems were identified in the literature search. Also, 17 anticholinergic lists were identified, which covered 278 medicines, of which 21 appeared on at least eight of the lists. In the empirical study, the women's (n = 745) mean (± SD) age was 83.35 (± 9.99) years, and they were older than the men (n = 241, mean age ± SD 75.11 ± 11.48, p < 0.001). The 1004 patients (response rate 70 %) were using a mean (± SD) number of 7.1 ± 3.4 regular medicines (range 0-20). 455 patients used no anticholinergics, 363 had some anticholinergic burden (score 1 or 2), and 186 had a high burden, with anticholinergic scores of 3 or more. The mean ARS score (± SD) was 1.2 ± 1.5 (range 0-10). When three anticholinergic lists were compared, all three lists identified only 280/791 of patients who were anticholinergic users according to at least one list. No association was found between anticholinergic medicine use and mortality. There are several methods available for measuring anticholinergic burden, but there is a need for a consensus method. This was highlighted by the lack of agreement on medicines on different lists and when three anticholinergic lists tested identified different patients when compared to each other. Anticholinergic use was common in this frail, older patient sample, but no effect on mortality was shown in this study setting. The cross-sectional nature of the data limits the reliability of the study, and any conclusions beyond older patients in Helsinki area must be done very cautiously. Future research should define anticholinergics better and investigate their possible effect on mortality in a prospective, randomised, and controlled setting.
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(2021)Antidepressant use among children and adolescents has become more common in many countries. The prevalence of antidepressants is higher for boys but during adolescence girls’ have a higher antidepressant prevalence. In previous studies, the prevalence of selective serotonin re-uptake inhibitors (SSRI) has increased. The aim of this study was to investigate antidepressant use among Finnish children and adolescents aged 1–17 years during 2008–2019. The differences of antidepressant use in different age groups and genders were investigated. Furthermore, the secondary objective was to examine the trends in prevalence and costs of the five most commonly used antidepressant agents. This was a nation-wide register study. The data for this study was from Kelasto which is a statistical database maintained by the Social Insurance Institution of Finland. The extracted data was from 2008–2018 and included each persons’ age, gender, dispensed drug and costs. The data extracted was for 1–17-year-olds who had been dispensed reimbursed antidepressants from community pharmacies. The data was analyzed with Microsoft Office’s Excel program. The results were transferred in to tables and reported as prevalences by age groups, genders, antidepressants and costs. The prevalence of antidepressant use among children and adolescents was 5,0 per 1000 in 2008 and it increased to 10,3 by 2018. In the youngest age group of 1–6-year-olds, antidepressant use decreased. Antidepressant use increased slightly among 7–12-year-olds. Antidepressant use increased the most among 13–17-year-olds. 13–17-year-old girls had the higher antidepressant use prevalence throughout the study. The same group had a 2,4-fold increase in prevalence during the study period which accounted for the biggest increase in the study. The most used group of antidepressants was SSRIs. The total cost for antidepressants among children and adolescents increased by 73,7 % during the study period. The most commonly used antidepressant agents were fluoxetine, sertraline, escitalopram, mirtazapine, and venlafaxine, respectively. Fluoxetine was the most used agent throughout the study. In 2014, sertraline surpassed escitalopram and became the second most used antidepressant agent. Escitalopram and venlafaxine’s cost per user decreased during the study. The cost per user stayed stable for mirtazapine. Fluoxetine and sertraline’s cost per user increased. The Kelasto database does not include data on indications for prescriptions. The prevalence of antidepressants does not necessarily correlate directly to depression among children and adolescents because antidepressants can be used to treat other diseases. More studies need to be conducted on different off-label uses for antidepressants among children and adolescents. This study only investigated the trends on cost for the five most commonly used antidepressants. Further studies on antidepressant costs among children and adolescents are needed. Additionally, it is essential to investigate the reasons for the increase in antidepressant use among children and adolescents.
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(2022)Multiple Sclerosis (MS) is an incurable autoimmune demyelinating disease affecting the central nervous system (CNS). Although the detailed pathogenesis remains unclear, recent research has highlighted the involvement of B cells. For decades, however, MS research was based on T cell-focused animal models of autoimmune encephalomyelitis (EAE), which do not reflect the involvement of B cells in the pathogenesis. Therefore, B cell-dependent EAE models are hypothesized to allow a better understanding of MS immunohistopathology and may therefore lead to the development of efficient treatments. In our spontaneous relapsing-remitting (RR) EAE model, B cells are recruited from the endogenous repertoire by transgenic myelin oligodendrocyte glycoprotein (MOG) -reactive T cells, causing the development of EAE in 3–4-month-old mice. Interestingly, MOG-specific antibodies are present long before actual onset of clinical disease and can be detected already in 5-week-old RR mice and disease development in RR mice is dependent both on the presence of (presumably MOG-specific) B cells as well as on stimuli provided by intestinal microbiota. Firstly, we evaluated the broader usability of induced germinal center cell (iGB) culture as a model for B cell repertoire studies. Then, by using iGB culture, we studied whether MOG-specific B cells are present in secondary lymphoid organs of younger than 4-week-old and germ-free RR mice. Finally, this study aimed to investigate whether the repertoire of MOG-specific B cells undergoes significant qualitative changes from young healthy mice to older acutely sick RR mice, and whether at the time of disease onset the recruited MOG-specific B cells expand and mature in the cervical nodes (cLN) or in the CNS. To do so, following the hosting-lab’s previous single-cell RNA sequencing (scRNA-seq) of B cells derived from cLN of 5-week-old RR mice, we performed the scRNA-seq of B cells from CNS, spleen, and cLN of acutely sick RR EAE mice. We demonstrated that iGB culture is an unsuitable tool to expand pre-activated B cells, and hence, in our hands it was inappropriate for repertoire studies. However, iGB culture proved to be useful for screening different organs for MOG-specific B lymphocytes, and we found that anti-MOG antibodies were firstly detected in 3-4-week-old RR mice, and MOG-specific B cells were present also in germ-free RR mice. Our scRNA-seq results revealed many highly expanded MOG-specific B cell clonotypes in acutely sick RR mice. Moreover, the B cell repertoire of sick RR mice was more diverse, including IgG1, IgM, IgG2b, IgG2c, and IgG3 isotypes, compared to healthy 5-week-old RR mice that had only IgG1 or IgM isotypes. Two-thirds of the expanded clonotypes were primarily detected in the CNS in sick RR mice, indicating that clonotypes develop further and continue isotype switching within the CNS. We also detected more somatic mutation in the variable region of expanded clones of sick RR mice compared to 5-week-old RR mice. The results of this study clearly show an antigen-driven evolution of the MOG-specific B cell repertoire from healthy young to acutely sick RR mice, which seems to occur mainly in CNS itself. In contrast, cLN are the major initial priming site of MOG-specific B cells in healthy RR mice, even under germ-free conditions. This suggests that commensal microbiota is not required for initial recruitment of MOG-specific B cells, but for the development of EAE. To further validate our encouraging scRNA-Seq results, it is necessary, in future experiments, to confirm the MOG-specificity of expanded clonotypes.
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(2016)Chlamydia pneumoniae is an intracellular bacterium that causes a variety of respiratory infections to humans such as pneumonia and bronchitis. In addition C. pneumoniae -infection has been associated with multiple chronic diseases of which the most important are atherosclerosis and vascular diseases, asthma, chronic obstructive pulmonary disease and different kinds of neurological disorders. C. pneumoniae is a very common pathogen that has the ability to hide in the system in a persistent chronic form out of reach of the immune defences. C. pneumoniae has been shown to infect many other cell types besides bronchial epithelial cells. These cells include monocytes, macrophages and vascular endothelial cells. C. pneumoniae induces the secretion of different kinds of cytokines and cell signalling molecules and the expression of adhesion molecules in all of these cell types. Too strong cytokine and immune response is detrimental to cells and to whole system. Currently available antibiotics aren't effective enough against C. pneumoniae -infection, especially against its chronic form. Furthermore, the lack of effective anti-chlamydial drugs impairs the research of the association between C. pneumoniae and chronic diseases. The aim of this study was to investigate the effect of anti-chlamydial compounds on the release of cytokines and cell signaling molecule, nitric oxide, induced by C. pneumoniae -infection in different cell types. These anti-chlamydial compounds are currently under the investigation in the faculty of pharmacy. In addition the anti-inflammatory properties of the compounds were further investigated with the help of lipopolysaccharide of another gram-negative bacterium E. coli. The groups of compounds investigated in this study were β2,2-amino acid derivatives, Schisandra chinensis -lignans, TE-compounds synthesized in Vienna and benzimidazole compounds synthesized in the faculty of pharmacy. There were four cell types used in this study, HL- and BEAS-2B-epithelial cells, THP-1-monocytes/macrophages and RAW264.7-macrophages. The study focused on the determination of vascular endothelial growth factor VEGF and interleukins IL-8, IL-10 and IL-12. The concentrations of cytokines in the cell medium were measured after infection using ELISA-method. Nitric oxide measurements were also determined from the medium using Griess' reagent. Immunofluorescence labeling was used to confirm the infection and the infection was verified by fluorescence microscope. In addition some of the compounds were tested for the cell viability using resazurin assay. All the groups of compounds showed desired effects on the release of cytokines and nitric oxide. Especially β2,2-amino acid derivatives reduced clearly the release of both cytokines and nitric oxide. β2,2-amino acid derivatives could thus be potential drug candidates for the development of anti-chlamydial and anti-inflammatory drugs. Schisandra chinensis -lignans inhibited especially the release of nitric oxide in both C. pneumoniae -infected and LPS-stimulated cells which may tell about their broad anti-inflammatory properties. There were also found desired results with TE-compounds and benzimidazole compounds. Interleukins were not secreted by any of the studied cells so that part needs more research and further investigation. Based on the results found in this study it can be concluded that the studied compounds could be potential lead compounds in the discovery of anti-chlamydial drugs and drugs that specifically inhibit C. pneumoniae -infection. Further research is needed concerning the effects of these compounds on cytokines and especially on chronic infection.
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(2024)Lääkityspoikkeamat aiheuttavat maailmanlaajuisesti eniten vältettävissä olevia potilashaittoja terveydenhuollossa. Vaaratapahtumista raportoiminen on tärkeää lääkitysturvallisuuden edistämiseksi, sillä raportoinnin avulla saadaan tietoa lääkehoitoprosessin riskikohdista, jota voidaan puolestaan hyödyntää kehitettäessä lääkehoitoprosessien suojauksia. Tutkimuksen tavoitteena oli selvittää, millaisia sosiaali- ja terveydenhuollossa tapahtuneita lääkehoidon vaaratapahtumia apteekeissa on havaittu ja estetty sekä millaisiin kehittämistoimenpiteisiin apteekkien vaaratapahtumailmoitukset johtivat tapahtumayksiköissä. Tutkimus toteutettiin retrospektiivisenä rekisteripohjaisena tutkimuksena avoapteekkien sosiaali- ja terveydenhuollon yksiköihin lähettämistä HaiPro-vaaratapahtumailmoituksista sekä tapahtumayksiköiden seurantalomakkeista. Tutkimusaineisto oli kerätty Pohjanmaan, Kanta-Hämeen ja Keski-Uudenmaan hyvinvointialueilla 1.2.2022-31.12.2023. Alkuperäinen aineisto sisälsi 457 vaaratapahtumailmoitusta. Aineiston esikäsittelyssä poistettiin ilmoitukset, jotka eivät olleet sosiaali- ja terveydenhuollossa tapahtuneita vaaratapahtumia (n=11). Ilmoitukset, jotka koskivat useampaa kuin yhtä lääkeainetta tai potilasta (n=15) jaettiin erillisiksi tapauksiksi. Strukturoitujen kohtien valintojen oikeellisuus tarkistettiin tapahtumakuvausten perusteella ja tarvittaessa valinta korjattiin. Lopulliselle tutkimusaineistolle (n=461) suoritettiin kuvaileva määrällinen analyysi (frekvenssit ja prosenttiosuudet) Microsoft Excel ohjelmistolla. Aineistosta laskettiin vaaratapahtumien määrä, luonne, havaitsija, tyyppi sekä asiakkaalle ja apteekille aiheutuneet seuraukset, yleisimmin esiintyneet lääkeaineet, lääkeaineryhmät sekä suuren riskin lääkkeiden osuus. Terveydenhuollon yksiköiden kehittämistoimenpiteille suoritettiin laadullinen sisällönanalyysi aineistolähtöisesti sekä tarkasteltiin kehittämistoimenpiteiden jakautumista yksilö- ja järjestelmänäkökulmiin. Lähes kaikki (94 %) aineiston vaaratapahtumista (n=461) oli apteekin havaitsemia. Valtaosa tapauksista oli läheltä piti -tapahtumia (71 %). Vaaratapahtumat olivat lähes aina lääkehoitoon liittyviä (98 %). Yleisimmin kyseessä oli määräyspoikkeama (93 %), jossa oli väärä annos tai vahvuus (26 %), epäselvä tai puutteellinen annosohje (13 %) tai SIC-merkintä puuttui (11 %). Eniten vaaratapahtumailmoituksia oli hermostoon vaikuttavista lääkeaineista (23 %) sekä systeemisesti vaikuttavista infektiolääkkeistä (19 %). Suurin osa sosiaali- ja terveydenhuollossa ehdotetuista kehittämistoimenpiteistä (n=470) oli tapahtuman käsittelyä ja siitä keskustelua (63 %). Yleisimmin ehdotettiin asian käsittelyä tai siitä keskustelua ylilääkärin ja lääkäreiden kanssa (15 %) tai moniammatillisessa palaverissa (14 %). Tämä tutkimus osoittaa apteekin roolin keskeisen merkityksen sosiaali- ja terveydenhuollon toimijana ja lääkitysturvallisuuden varmistajana. Tutkimuksen perusteella apteekkien vaaratapahtumailmoittaminen voi tukea muun sosiaali- ja terveydenhuollon lääkehoitoprosessien turvallisuuden kehittämistä, mutta kehittämistoimien vaikuttavuutta lääkehoidon vaaratapahtumien ilmaantumiseen tulisi tutkia lisää.
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(2023)Medication safety is an important target of development in health and social services systems internationally. Medication errors are one of the biggest risk factors in medication safety. Majority of the medication incidents could be avoided by improving the medication treatment process. Patient safety incident reporting systems enable health and social services to collect systematic data from risk factors within the medication treatment process. This study was conducted as a retrospective registry-based study where medication incidents that occurred in health and social care units reported by community pharmacies to the incident reporting system HaiPro from 21st of September 2021 to 31st of October 2022 were analysed. Cases that did not meet the criteria for this study (n=55) were removed from the original data (n=3841). If needed, the nature and type of the reported error were corrected. A descriptive quantitative analysis was conducted for the final data (n=3786) using Microsoft Excel. The number, natures, types, observers, and prescription types of medication errors were investigated from the data. In addition, the most common groups of medicinal substance and high risk medicines were identified. A qualitive content analysis was performed to near miss cases involving high-risk medications (n=446) using the Atlas.ti program. Interventions, measures following the interventions and risks prevented by the measures were identified from the open description in the incident reports. The qualitative analysis was performed as an abductive content analysis. Of the medication errors included in the study (n=3786) 91% were detected by community pharmacies and the majority (68%) of the reported incidents were near misses. Most (96%) of the safety incidents (n=3786) were associated with the patient’s medication treatment and had occurred mostly during the prescribing process (92%). As a result from the prescribing errors, patients were most commonly prescribed wrong dose or strength of the medicine (26%) or the prescription lacked SIC marking (26%). High-risk medications occurred in 16% (n=591) of the incidents (n=3786). Most frequently detected high-risk medications were opioids (35 %). Three quarters (76 %) of safety incidents associated with high-risk medications were near misses (n=446). The majority (92 %) of interventions (n=471) made to prevent safety incidents associated with high-risk medications were made by community pharmacies. The most frequent intervention was community pharmacies contacting the doctor. Based on the HaiPro incident reports made about medication errors in health and social care units reported by community pharmacies, it can be concluded that community pharmacies are a central barrier in primary care medication treatment process. Community pharmacies detect and report medication errors that have occurred in other health and social care units. Safety incidents reported by pharmacies systematically accumulate important information that can be used in the development of medication safety in primary care at a unit, wellbeing services county and national levels.
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(2022)The operation of community pharmacies has developed extensively over the past decades, with special emphasis on medication counselling services. In addition to dispensing, pharmacies can offer various kinds of clinical pharmacy services, such as medication reviews, automated dose dispensing and other services to support rational use of medicines. All this activity requires patient information, which is currently available in pharmacies only from prescriptions, reimbursement information, and by asking the customer. Because of this, a need to increase the availability of patient information in pharmacies has come up. The aim of this study was to determine what kind of patient information should be available in community pharmacies for 1) the statutory dispensing of medicines, the medication counselling and treatment monitoring, and 2) other services related to promotion of health and well-being and prevention of diseases. Furthermore, the study investigated experts' experiences of the sufficiency of patient information in pharmacies, as well as in what form and from what period the information should be available in pharmacies. The study was conducted as a 3-round Delphi study with an expert panel consisting of 20 pharmacists specialized in clinical pharmacy. Consensus was formed with the help of a preliminary patient information list which had been compiled based on the literature and the expertise of the research group (a total of 39 patient data items). The limit of the experts' consensus was set to ≥80%. The Delphi-rounds were conducted as electronic surveys during the spring and summer of 2022. The responses were analysed using quantitative and qualitative methods. Most of the expert panellists (n=20) perceived that the patient information available in community pharmacies was insufficient. This study reached a strong consensus that pharmacies should have quite a large set of patient information available both for dispensing medicines and medication counselling, and for providing services supporting rational use of medicines. Of the patient data items, nine reached the consensus line concerning dispensing of medicines and 31 measures concerning other services. From both points of view, information about the client's diagnoses, blood pressure, and the GFR value indicating kidney function were rated as the most important to be available in community pharmacies. However, the panellists also reported challenges to overcome in the access and utilization of the patient information, for example, related to current legislation, resources, and competences of pharmacists. These aspects should be considered in the development of community pharmacy practice and electronic patient information (e.g., Kanta services).
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