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  • Holma, Paula (2011)
    Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.
  • Silén, Jenna (2021)
    The life cycle of Chlamydia pneumoniae is a biphasic developmental cycle, as a obligate intracellular bacterium, it forms various morphological forms, including elementary bodies, reticulate bodies and aberrant bodies belonging to a persistent form. Due to the bacterial life cycle and the fact that chronication of C. pneumoniae infection and formation of persistent infection as well as pathogenesis is a complex problem involving multiple signaling pathways and affecting several different cells, it is useful to seek medication to influence infection from different stages of the bacterial life cycle. There are several different factors that induce persistence and thus models of persistence. Although the detection of aberrant RBs and thus aberrant bodies in C. pneumoniae infected tissues does not provide complete certainty about chronic infection, the bacterium has been linked to chronic health problems such as atherosclerotic cardiovascular disease and asthma. The aim of the study was to develop a persistence model induced by beta-lactam antibiotics, amoxicillin and penicillin G, in A549 cells by monitoring the size, shape, and number of inclusions using the IPA method and the immunofluorescence staining method for infection. In addition, the antibiotic sensitizing effect of three compounds on pulmonary chlamydial infection was studied. This effect was monitored by examining the recovery of persistent infection and by monitoring the protective effect of the compounds on beta-lactam-induced persistence. The work succeeded in finding an infection model that is well suited for studying beta-lactam persistence. Due to treatment recommendations, pulmonary chlamydial infections are practically treated with beta-lactam antibiotics. Based on the methods used, it was found that amoxicillin concentrations of 10 and 25 µg/ml and penicillin G concentrations of 100 U/ml and 250 U/ml were sufficiently effective to transfer bacteria to a state of persistence. It was found that the amoxicillin persistence model is reversible based on the increase in the size of the inclusions, especially at 25 µg/ml and quantitatively at 10 µg/ml. It was concluded that amoxicillin at a concentration of 10 µg/ml is sufficient to induce persistence in a beta-lactam antibiotic-induced persistence model. Further quantitative studies on the persistence model are needed, such as quantitative PCR based on the OmpA gene to determine more accurate dose-response relationships. Glutathione levels should also be monitored in the persistence model.
  • Pietarinen, Teemu (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).
  • Mikkonen, Heidi (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.
  • Kiuru, Karoliina (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.
  • Peltola, Roosa (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.
  • Selin, Markus (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.
  • Takala, Hanna-Elina (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.
  • Kainulainen, Saila (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.
  • Rautiainen, Swarna (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.
  • Eronen, Sini-Tuulia (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.
  • Kovanen, Satu (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.
  • Kumpula, Eeva-Katri (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.
  • Hou, Kathy (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.
  • Hämäläinen, Klaus (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.
  • Lohtaja, Milka (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.
  • Laakso, Johanna (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).
  • Luhtanen, Päivi (2020)
    The pharmacy operations are strictly regulated in Finland and the operation of a pharmacy business requires a licence. Number of community pharmacies has stayed quite steady for the past 10 years. At the end of the year 2019 there were 817 pharmacies or their subsidiaries in Finland. The number of pharmacies is expected to increase, since 29 new pharmacies has been established since 2016. The inspection of pharmacies is a part of the legal duties of the Finnish Medicines Agency. In Finland, pharmacies are inspected based on a risk assessment, as often as it is necessary to ensure appropriate operations of a pharmacy. During a pharmacy inspection, the focus is on operations that are critical to drug safety and medication safety. The aim of an inspection is to make sure that pharmacy operations comply with the regulations. There are only few studies made on remote inspection of the pharmacies. The Finnish Medicines Agency hasn’t made remote pharmacy inspections before. The aim of this study was to develop, validate and test a questionnaire, which could be used to inspect community pharmacies remotely and to develop and test a process for remote inspections. The study was done in two parts. In the first part, a draft of the questionnaire was developed by studying the regulations and laws regulating the operations of a pharmacy and by using a content analysis. The material for the content analysis was a pre-inspection questionnaire form, answers to the pre-inspection questionnaires and defect lists of the inspection reports of those pharmacies (n=37), which had answered to the pre-inspection questionnaire before pharmacy inspection in 2019. Content of the pre-inspection questionnaire and the answers of pre-inspection questionnaire were compared to the content of the defect lists of inspection reports. The aim of the comparison was to find out how the existing pre-inspection questionnaire could be utilized when developing the questionnaire for the remote pharmacy inspections. In addition, the listed defects of the inspection reports were categorized to explore what were the most common defects observed during pharmacy inspections. In the second part of the study, the content of the developed questionnaire was validated by using a three round modified Delphi survey. Seven experts with good knowledge of the pharmacy inspections were chosen to the Delphi panel. The aim of the Delphi rounds was to achieve full consensus among the experts about the content of the questionnaire. Alongside the Delphi rounds, a process to remote inspect a pharmacy was developed. The remote inspection questionnaire and the process were tested internally in the Finnish Medicines Agency at the end of the second stage of study. The draft of the questionnaire included 15 sections and 164 questions. Based on the comments received during the Delphi rounds, the content of the questionnaire was modified. On the third Delphi round a full consensus of the content of the questionnaire was achieved among the experts. The final questionnaire for the pharmacy remote inspection included 14 sections and 184 questions. The process of the remote pharmacy inspection follows the procedure of an on-site pharmacy inspection. In the internal test, the process of the remote inspection was found to be a good way to inspect pharmacies remotely. The remote inspection process is a new way to inspect pharmacies. With the remote inspection, it is possible to find out the most common defects on the pharmacy operations by using the questionnaire and contact calls. The remote inspection questionnaire and the process need to be further tested to ensure that the process is optimal from the perspective of the authority and the pharmacies.
  • Saksi, Outi (2016)
    The development and maintenance of the pharmaceutical workforce's know-how ensure the availability of medicinal consulting and service. Healthcare personnel in Finland are bound by law to uphold and improve their workmanship. Furthermore, a pharmacy owner is legally obliged to keep track of the development of healthcare professional's skills and to ensure the staff's sufficient participation for continuing education (CE). Pharmacists' development and maintenance of professional skills is not linked to preservation of professional competence in Finland. The goal of this study was to get a general view of the development and maintenance of professional skills of pharmacists working in community pharmacies as well as applicaple methods. Additionally, the aim was to determine whether community pharmacists' development of professional skills is systematical. As background material in this thesis, a sub-material of an online study regarding development and maintenance of professional skills was used, which was carried out by the Finnish Pharmacists' Association in September 2013 and it consists of 430 pharmacists' responses who work in community pharmacies. The results show that the methods community pharmacists use to develop and maintain their professional skills are diverse. The recommendation by the authorities is at least three days of CE for one person per year but the majority (83 %) of the participants of this study didn't follow it. Some of the pharmacists develop and maintain their professional skills by attending diligently CE's while the number of pharmacists who do not attend any CE has risen. The number of pharmacists who did not participate in any CE was 26 % in the year prior to this study. The results might point to changes in learning methods or the decline of CE activity. The results of this thesis show that development of professional skills was not systematical in the majority of the pharmacies. An annual personal develompent plan was drawn up in 24 % of the respondents' workplaces and development discussions were had in few. Independent planning, monitoring and evaluation of their own professional skill development were done by 10 % of the pharmacists. The planning of professional skill development was not found to impact CE participation. Development discussions and training schedules that are drawn up in workplaces were found to increase pharmacists' independent planning of their professional skills.
  • Mäkinen, Emilia (2021)
    Background and objectives: Documenting and processing of dispensing errors at both organizational and national levels is one of the basic preconditions for effective medication risk management. Since the most recently accomplished national register research of dispensing errors in Finland, there have been several changes in the medication dispensing process that advance medication safety. Thus, the previous study does not provide an up-to-date picture of the current situation. The primary objective of this study was to find out the trends in dispensing errors that were reported to the Finnish Pharmacy Association's registry of dispensing errors in 2015–2019. The secondary objective was to identify risk factors expository to dispensing errors in the dispensing process and to review the measures utilized by community pharmacies to prevent dispensing errors. Materials and methods: The retrospective registry study, in which the register of dispensing errors maintained by the Finnish Association of Pharmacists for the period from 1 January 2015 to 31 December 2019, was analyzed. Cases that did not fulfil the definition of a dispensing error (n=829) were removed from the original data (n=17763). In addition, clear errors (n=2130) were corrected in the data and cases (n=499) that were initially insufficiently entered in the register were added. 17433 dispensing errors were included in the study. The data was analyzed using Microsoft Excel. The number, qualities, prescription types, observers, therapeutic harms and contributory factors of the dispensing errors were investigated in the data. The most common groups of medicinal substance, high-alert medications and risk factors in the medication dispensing process were identified in the data. In addition, interventions reported by community pharmacies to prevent dispensing errors were collected from the data. Results: The number of cases reported to the dispensing error register has decreased annually (2015 n=3913, 2016 n=3795, 2017 n=3708, 2018 n=3578, 2019 n=2439). The most common types of dispensing errors are incorrect strength (51 % of all the reported dispensing errors) and incorrect quantity or package size (14 %). Slightly more than a half (51 %) of the reported dispensing errors were noticed by medicine users. The percentage of electronic prescriptions in dispensing errors has increased and is clearly the most common prescription type in dispensing errors (2015: 79 %, 2016: 84 %, 2017: 93 %, 2018: 96 %, 2019: 95 %). The majority of dispensing errors occurred with cardiovascular medicines (29 %) and medicines affecting the nervous system (26 %). 7 % of dispensing errors caused therapeutic harm to the medicine user. As a result of dispensing errors, 21 medicine users were hospitalized. 13 % of dispensing errors occurred with high-alert medications (n=2244). The high-alert medications were involved in one-third (n=7) of dispensing errors that led to hospitalization. Factors related to the employee (25 %), similar packaging (19 %), and similar medicine name (15%) were most commonly considered to be the main contributory factors for the occurrence of the dispensing errors. The risk factors identified in the medicine dispensing process were related to the pharmacy system, the characteristics of the prescription, the storage method of the medicine and the characteristics of the medicine packaging. In the automated dose dispensing process, the risk of dispensing error increased if changes had to be made to the dose dispensing order. The risk factors for automated dose dispensing were related to the pharmacy system and the characteristics of the prescription. The community pharmacies had mentioned taking measures to prevent dispensing errors in one-fifth (21 %) of the reported cases. In addition to developing their own operations, community pharmacies saw cooperation with other healthcare professionals as an important factor in preventing medication errors. In addition, community pharmacies reported exposing properties for dispensing errors of pharmaceutical products and systems to pharmaceutical companies and providers of pharmacy systems and automated dose dispensing. Conclusions: Trends, risk factors of the dispensing process and interventions to prevent dispensing errors can be identified in the dispensing errors reported to the Finnish Association of Pharmacists’ dispensing error registry. The dispensing error register provides valuable information on dispensing errors at the national level, but it is no longer able to fulfil completely the current medication safety needs. In the future, the role of the pharmacy as a promoter of medication safety should be perceived as more comprehensive. In the development of medication safety, special attention should be paid to the risk factors of the dispensing process, the high-alert medications and to new risks arising from the increase of electronic prescriptions and automated dose dispensing. In addition, cooperation between pharmacies and other healthcare professionals and the medication safety culture of pharmacies should be further strengthened.