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(2017)Left ventricular hypertrophy (LVH) is defined as an increase in left ventricular mass. It is initially a coping mechanism by which the heart tries to compensate for the increase in load caused by, for example, hypertension, but it will eventually lead to heart failure. LVH is the result of primarily an increase in cardiac myocyte size, in addition to increased apoptosis and necrosis of cardiac myocytes and fibrosis. Current treatment of LVH is based on a treatment of suspected cause, generally hypertension. Antihypertensive medication has been found to have beneficial effects on LVH. However, antihypertensive drugs can not cure LVH completely, hence other treatment options are needed. To identify new possible drug targets, it is important to increase the inadequate knowledge of the mechanisms and signal transduction pathways mediating LVH. The most relevant stimuli causing hypertrophy are considered to be mechanical stretch, as well as some humoral mediators such as angiotensin II and endothelin 1 (ET-1), to which cardiomyocytes respond through activation of several intracellular signal transduction pathways. As a result, cardiomyocyte gene expression and protein synthesis increase and sarcomeres grow and rearrange, resulting in an increase in cell size. In addition, regulation of calcium, contractile function and energy metabolism of cardiac myocytes change. Numerous intracellular signal mediators interact with each other and can compensate for each other, making it difficult to investigate the significance of individual factors. As important signal mediators are considered to include protein kinase C (PKC) and cardiac transcription factors GATA4 and NKX2-5. In vitro studies of cardiac hypertrophy are usually performed with primary cardiac myocytes isolated from the ventricles of neonatal rats. The H9c2 continuous cell line has been used in some studies as an alternative cell model to reduce the use of laboratory animals. In the experimental part of this thesis, the suitability of H9c2 cells for hypertrophy studies was examined by comparing them to primary cardiac myocytes. In addition, experimental compounds targeted to cardiac transcription factors and PKC were studied by exploring their effects on viability and hypertrophic responses of H9c2 cells and primary cardiac myocytes. The toxicity of the compounds and the effects on cell viability were studied using the lactate dehydrogenase (LDH) assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The hypertrophy responses to cyclic mechanical stretch and ET-1 were primarily assessed by measuring the surface area of cells from fluorescence microscopy images. In addition, the relative expression levels of Nppa and Nppb genes in ET-1 stimulated primary cardiac myocytes were studied by quantitative polymerase chain reaction (qPCR). Both stretching and ET-1 caused an increase in the cell surface area in primary cardiac myocytes but not in H9c2 cells. On this basis, the H9c2 cells respond differently to hypertrophic stimuli than primary cardiac myocytes, and the suitability of H9c2 cell line to hypertrophy studies can therefore be questioned. The compounds targeted to cardiac transcription factors were not cytotoxic at 1-30 µM concentrations, but they also had no significant effect on the hypertrophic responses. In contrast, the PKC compound HMI-1a3 at 30 µM was toxic to primary cardiac myocytes and HMI-1b11 at 30 µM was toxic to H9c2 cells. HMI-1b11 and bryostatin-1 also induced changes in the hypertrophic responses of primary cardiac myocytes, but the significance of these results requires further investigation.
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(2014)The impact of nanoparticulate drug carriers, especially polymeric micelles, is growing continuously. However, their drug delivery properties in vivo are difficult to predict. In this thesis, the approach of screening a combinatorial library of nano carriers for their drug delivery properties in a high throughput/high content (HT/HC) manner was tested. The library consisted of self-assembling polymeric micelles, using the amphiphilic polymer DSPE-PEG2000. The physicochemical characterization of micelles was focused on size, shape and stability, tested by various methodologies. The micelles were labeled with the fluorescence dye Alexa568 and the combinatorial character was based on labeling with two different Cell Penetrating Peptides (CPP), RGD and transactivator of transcription (TAT), in three molar ratios each. The cytotoxicity concentration ranges and micelle uptake were tested in the ARPE-19 cell line. Intracellular localization was observed by confocal fluorescence microscopy. Quantitative HCS imaging analysis was performed by image cytometry, whereas only the parameter 'micelles spots per cell' was analyzed exemplarily. The quantitative HCS results were not clear and pointed to insufficient optimization of experimental and analytical parameters. The results suggest that HCS could be a suitable method to analyze a nanoparticulate library for its drug delivery properties, requiring careful optimization of experimental parameters. However, the careful characterization of the micellar library is a critical factor in planning and understanding biological experiments.
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(2023)Orexins (hypocretins) are two neuropeptides, orexin-A (OX-A) and orexin-B (OX-B), produced by a neuron subpopulation in the mammalian hypothalamus. They are natural substrates of two G-protein-coupled receptors (GPCRs), orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R), respectively. Orexin receptors are expressed widely in the central nervous system. Orexin peptides and receptors were originally discovered in 1998 and have been extensively researched ever since. Maintaining a steady state of wakefulness has been identified as one main physiological function of orexinergic signalling, and loss of orexinergic neurons in the hypothalamus has been linked to narcolepsy. Over the past decade orexin receptor antagonists have been developed for treatment of insomnia – suvorexant as the first one, approved for clinical use in 2014. Orexin receptor agonists remain under development for treatment of narcolepsy as one potential therapeutical indication, with no clinical applications yet approved. Orexin receptor activation by small-molecule agonists has proven a challenge not yet conclusively resolved. The aim of this study was to validate a novel scaffold for orexin receptor agonists from compounds identified as orexin receptor agonists in previous studies. Total of ten compounds were designed for synthesis, three of which were successfully synthesized. These three compounds exhibited very low orexinergic activity (0.06–1.36% and 2.33–5.19% response of full activation for OX1R and OX2R, respectively). After activity testing retrospective modelling of the receptor binding of the synthesized and designed compounds was implemented computationally by structure-based molecular docking to the recently discovered (2021) crystal structure of OX2R in complex with bound small-molecule agonist 3′-(N-(3-(2-(2-(2H-1,2,3-triazol-2-yl)benzamido)ethyl)phenyl)sulfamoyl)-4′-methoxy-N,N-dimethyl-[1,1′-biphenyl]-3-carboxamide. Some of the key interactions known as crucial for receptor activation, such as hydrogen bonds with glutamine Q1343.32, were found possible for some of the synthesized and designed compounds. This may in part explain the orexinergic activity, however very low, measured for the synthesized compounds. Low activity of the synthesized compounds may be a result of low binding since their binding was not tested in this study. Interactions between the synthesized compounds and OX2R predicted by molecular modelling are consistent with the low measured activity of the compounds, and alternative, more optimal chemical scaffolds for orexin receptor activation could be searched in future studies.
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(2016)The aim of this study was to synthesize antimicrobial and anti-biofilm agents using abietic (AA) and dehydroabietic acids (DHAA). Bacterial biofilms are formed when bacteria cells cluster together within a self-produced extracellular matrix. This lifestyle makes bacteria highly resilient to different environmental stresses and conventional antibiotics when compared to single-cell bacteria. Currently, there are no approved anti-biofilm agents as drugs and only a few number of compounds can selectively target biofilms and eradicate them at low concentrations. Potent drugs targeting them are needed. AA and DHAA are abietane-type diterpenoids found in the resin of conifer trees. Antibacterial effects of resin acids have been widely studied, specifically against methicillin-resistant Staphylococcus aureus strain (MRSA). Through the combination of DHAA with different amino acids, Manner et al. (2015) discovered a new class of hybrid compounds that target both planktonic and biofilm bacteria in Staphylococcus aureus. The study group also discovered two of the most potent abietane-type anti-biofilm agents reported so far in literature. This thesis followed the work of the research group by designing and synthesizing additional AA and DHAA derivatives to target bacterial biofilms. Rings A, B and C of the diterpenoid core were modified and 24 derivatives were successfully synthesized. Amino acids were attached to the compounds either before or after ring modification. Standard structural elucidation techniques were used to confirm the structure of the synthesized compounds.
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(2021)Prolyl oligopeptidase (PREP) is a serine protease that is widely found throughout the human body and especially in the brain. The primary function of PREP is thought to be the hydrolysis of the carboxyl side bond of proline residues in oligopeptides. PREP is also shown to increase the dimerization and aggregation of α-synuclein and downregulate the protein phosphatase 2A mediated autophagy in the cell via direct protein-protein interactions (PPI). The accumulation of α-synuclein aggregates in cell is known to cause α-synucleinopathies such as Parkinson’s disease. This makes the PPIs of PREP an attractive target for drug research. The mechanisms of the PPIs of PREP are still poorly understood. Recent studies have shown that these PPIs can be modulated with ligands lacking high inhibitory activity for the proteolytic activity. These studies show that the IC50-value of the ligand does not correlate with ligands ability to affect the PPIs of PREP. Ligands that could selectively modulate the PPIs of PREP without inhibiting the proteolytic activity of PREP could give valuable information on the mechanisms of the PPIs and on how to modulate them. It is hypothesized that the ligands could bind to PREP at a site that does not interfere with its proteolytic activity, and ligand binding is assumed to restrict the dynamic structure of PREP and thereby also modulating the PPIs of PREP. The aim of this study was to synthetize novel peptidic PREP ligands and study their effects on the proteolytic activity of PREP and the PPIs of PREP. The aim was to find and identify ligands and structures that would modulate the PPIs of PREP and observe how the IC50-values of the ligands would correlate. L-Alanyl-pyrrolidine was selected as the scaffold for the compound series and the five-membered heteroaromatics, imidazole, triazole and tetrazole, were added to the 2-position of the pyrrolidine ring. In this position there is an electrophilic group in many PREP inhibitors, although these heteroaromatics are not electrophiles. The scaffold was also expanded by adding phenyalkyl groups with different linker lengths were added to the N-terminal side of the alanine. The ligands were synthesized using four synthesis routes which were based on synthesis methods found in literature. The IC50-values and the effects on α-synuclein dimerization and autophagic flux were determined for five synthetized compounds. The tested compounds were all weak PREP inhibitors and showed no strong activity in the α-synuclein dimerization and autophagy assays. Despite the weak activities in the assays, the importance of the linker length in the phenyalkyl group was shown. Changing the linker by one methylene group had noticeable effect on the overall activity. The results also demonstrate a lack of correlation between the IC50-values and the effects on α-synuclein dimerization and autophagic flux, which further confirms the lack of correlation between the proteolytic function and the PPIs of PREP which was observed also in previous studies.
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(2024)Antimicrobial resistance (AMR) is a growing global health concern, and the development of new antibacterial agents is crucial to addressing this issue. Commercial antibiotics are not as effective as they used to be to combat infections. Previous studies have demonstrated the promising antimicrobial activity of etrasimod and one of its derivatives, compound 24f, against Gram-positive species. Therefore, as part of this study, we modified the carboxylic acid functional group to produce new derivatives. We synthesized derivatives of etrasimod and 24f, in order to generate a variety of compounds for evaluation of their antimicrobial effectiveness. Furthermore, the study evaluates the compounds' in vitro antibacterial activity and in vivo efficacy using Caenorhabditis elegans worms as an infection model. C. elegans is a widely used model organism in biological research, and it is particularly useful for studying host-pathogen interactions and drug efficacy. In addition, the cytotoxicity on mammalian cells (HeLa) was determined. Compound 18 showed the lowest cytotoxicity level (CC50 = 75.71±14.4 µM) of tested compounds. The antibacterial activity of new etrasimod derivatives was tested against Gram-positive (Staphylococcus aureus, including methicillin-resistant strains (MRSA)) and Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli). The tested compounds showed activity against Gram-positive bacteria but not against any of the Gram-negative strains. Compounds 9 and 18 showed to be the most active compounds, having a minimum inhibitory concentration of 5–6 and 8–10 μM, respectively. Moreover, both compounds showed promising activity in vivo, being able to significantly reduce the bacterial load in infected worms and improve their survival rates in survival experiments. The study provides insights into the development and assessment of potential antibacterial agents, addressing the contemporary challenge of AMR. The study's findings suggest that compounds 9 and 18 could be potential candidates for further development as novel antimicrobial agents.
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(2024)Since their discovery, β-lactam antibiotics have been an essential part of antibacterial chemotherapy. However, antibacterial resistance against β-lactam antibiotics via β-lactamases is a major challenge since it reduces the efficiency of current antibiotics. There are clinically available serine-β-lactamase (SBL) inhibitors, yet metallo-β-lactamase (MBL) mediated resistance is an arising research problem due to their ability to hydrolyze SBL-resistant carbapenems, and SBL inhibitors as well. N-sulfamoylpyrrole-2-carboxylates (NSPCs) are one of the MBL inhibitor chemotypes that displayed potent inhibitory activity against various subtypes of MBLs. This thesis focuses on the synthesis of novel NSPCs. Additional purification methods were applied in order to improve the current procedure. A large-scale synthesis of the starting material was conducted first. Thienyl, and halogen and methoxy substituted phenyl attached NSPC analogues were selected to be synthesized. However, the syntheses failed at the Suzuki coupling stage for the three compounds, and for the other two the purification of the final compounds was not successful. The thesis offers valuable insights into how various purification methods affect the yield at various stages of synthesis. Despite unexpected results, it also highlights the challenges in creating new NSPC analogues.
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(2019)Prolyl oligopeptidase (POP) is a serine peptidase found at high concentrations in the brain, which cleaves short proline-containing peptides at the carboxyl side of proline. POP activity has been shown to differ between healthy people and ones suffering from certain neurodegenerative diseases. Amongst its other functions, it has been shown to accelerate α-synuclein aggregation. Inhibiting the enzyme prevents this acceleration. Many highly potent peptidic POP inhibitors, based on the enzyme’s natural substrates, have been synthesized. One of the problems with many of these peptide-like inhibitors is their inability to penetrate the blood-brain barrier efficiently. Recently, a new surprisingly potent non-peptidic inhibitor based on a novel heteroaromatic scaffold was discovered. There was a need to synthesize analogues of this inhibitor in order to gain a better understanding of the structure-activity relationship for compounds based on the new scaffold. Unfortunately, the heteroaromatic scaffold is relatively unstable without a stabilizing substituent. The aim of this research was synthesizing stable analogues, primarily by replacing the original heteroaromatic ring with other, more stable heteroaromatic rings. It was hypothesized that the activity of the original compound could be retained, as heteroaromatics can often act as bioisosteres of each other. Multiple close analogues containing the new heteroaromatic rings were successfully synthesized and tested. Although they were considerable more stable compared to the original compound, there was a significant decrease in potency. However, the new compounds were not completely inactive, and they provided useful information on the viability and importance of several different substituents. Furthermore, measuring the IC50 value is not enough to evaluate their overall effect on POP, since the inhibition of the proteolytic activity of POP does not seem to correlate with the inhibition of its other functions. Further investigation and development of the new compound series is needed.
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(2010)The human tissue kallikreins (KLKs) form a family of 15 closely related serine proteases (KLK1-15). KLK3 is better known as prostate specific antigen (PSA) and it is highly prostate-specific. Kallikreins are attracting increased attention due to their role as biomarkers for screening, diagnosis, and monitoring of various cancers. Although PSA is a very useful marker for prostate cancer in the blood, the expression level of PSA is higher in normal prostatic epithelium than in tumour tissue, and it is further reduced in poorly differentiated tumours. It has been postulated that PSA activators (stimulating compounds) could be beneficial for patients with prostate cancer. The development of peptides as clinically useful drugs is greatly limited by their poor in vivo stability and low bioavailability. As the problems in using peptides as drugs are mainly arising from the peptide backbone, the focus for synthesizing peptide mimicking compounds is on the peptide backbone and how to replace it. The aim of this work was to replace the central disulfide bridge in the most potent PSA activating peptide C-4 by a hydrocarbon linker that had been previously synthesized in the research group. Two strategies for synthesis of the pseudopeptides were applied: a) synthesis of all peptide bonds on solid support by tailoring the reactions for this particular peptide, and b) synthesis of a monocyclic pseudopeptide in solution that could be incorporated directly into the standard protocol of solid phase peptide synthesis. The first strategy (a) proved to be tedious and would have required a lot of optimization to be successful. The cleavage conditions of the orthogonal protecting groups were not directly compatible with synthesis on solid support. The second strategy (b) also proved to be tedious, epimerization at the histidine residue was very prone in the solution phase even with standard peptide coupling reagents. However, the possibility to monitor all steps of the synthesis and to purify intermediate products made this synthetic route more attractive for this type of pseudopetide. The work in this master thesis resulted in a useful strategy to synthesise the desired pseudopeptides.
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(2015)Seas are one of the most biodiverse and species-rich areas on the planet. Many of the underwater species are yet to be found and identified. The marine based drug discovery and the clinical pipeline of marine compounds have increased lately. Thus, there is a strong believe that the marine-derived compounds will provide new pharmaceutical lead compounds. Marine sponges are one of the most studied marine species. Sponges can be found in shallow and deep waters all over the world. Pseudoceratina purpurea is a Verondiga order sponge that is known to be a source of bromotyramines. Bromotyramines are tyramine derivatives that have represented biological activity including cytotoxity, antivirality and antimicrobial effects. Purpurealidin E is a bromotyramine that has been identified from Pseudoceratina purpurea. Purpurealidin E hasn't showed remarkable biological activity by itself, but it can be used as starting point for synthesis of novel bromotyramine derivatives. By forming an amide bond between carboxylic acid and primary amine of purpurealidin E, new bromotyramines can be synthesized. In this master's thesis, purpurealidin E was successfully synthesized. Total amount of 11 novel bromotyramine derivatives were synthesized by amide coupling. Three of the new bromotyramine derivatives and purpurealidin E were purified and their biological activity against hepatitis C virus (HCV) was evaluated. Purpurealidin E did not show any antiviral activity, but all the three compounds showed potential biological activity against HCV. This work can be considered to a continuum to the now ended MAREX project (Exploring Marine Resources for Bioactive Compounds: From Discovery to Sustainable Production and Industrial Applications).
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(2024)Parkinson’s disease (PD) is a prevalent neurodegenerative disease characterized by movement disorders, such as bradykinesia, akinesia, and tremor. The degeneration of dopaminergic neurons in the central nervous system (CNS) is the most central aspect of the pathophysiology of PD-related movement disorders. The treatment of PD motor symptoms is based on increasing the diminished dopaminergic signalling in the CNS. This can be achieved by using medications such as dopamine agonists and monoamine oxidase B inhibitors. Levodopa, which acts as a precursor of dopamine in the body, is currently considered the most effective treatment for PD motor symptoms. Unlike dopamine, levodopa can cross the blood-brain barrier. Thus, levodopa must reach the CNS before being metabolized into dopamine to achieve the desired therapeutic effect. Dopa decarboxylase (DDC) inhibitors and catechol-O-methyltransferase inhibitors have been co-administered alongside levodopa to reduce its peripheral metabolism. However, when administered orally, levodopa is also metabolized in the gut by tyrosine decarboxylase, an enzyme produced by gut bacteria. Inhibi tion of bacterial tyrosine decarboxylase (TyrDC) could increase the effectiveness of levodopa treatment and reduce the needed levodopa dosage. The aim of this study was to synthesize and assess the biological activity of novel analogues of previously identified hit compounds which are dual inhibitors of TyrDC and DDC. Our goal was also to gain a deeper understanding of the structure-activity relationships of these compounds. Some of the compounds synthesized in this study were able to inhibit both TyrDC and DDC. Unfortunately, they were also either toxic towar ds human cells, and/or lacked efficacy in a bacterial cell-based assay used to determine the inhibition of levodopa metabolism. However, the data generated in this study can be utilized to design and synthesize new analogs to discover more efficacious and safer TyrDC and DDC dual inhibitors.
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(2023)Biologiset lääkkeet ovat keskeinen osa syöpäsairauksien hoitoa. Ne ovat usein perinteisiä pienimolekyylisiä lääkkeitä kalliimpia, ja niiden aiheuttamia kustannuksia potilaalle ja yhteiskunnalle voidaan hillitä edullisempien biosimilaarien eli vertailukelpoisten biologisten lääkkeiden käytöllä. Tutkimuksen tavoitteena oli tutkia kyselyaineiston perusteella syöpäpotilaiden näkemyksiä lääkärin toteuttamasta biologisten lääkkeiden lääkevaihdosta ja niihin vaikuttavia tekijöitä avoterveydenhuollossa. Lisäksi tutkittiin, miten yleisesti syöpäpotilaat tunnistavat biologisen lääkkeen ja biosimilaarin käsitteet, ja potilaiden syöpälääkitystään koskevia lääketiedon lähteitä. Tutkimuksen osana toteutettiin järjestelmälliseen kirjallisuushakuun perustuva katsaus syöpäpotilaiden näkemyksistä biologisista lääkkeistä ja/tai biosimilaareista ja niiden lääkevaihdosta. Tutkimus perustui Yliopiston Apteekin ja Helsingin yliopiston yhteistyössä Suomen Syöpäpotilaat ry:n kanssa toteuttamaan tutkimuskyselyyn syöpäpotilaille tammikuussa 2021. Tutkimuskutsu lähetettiin Yliopiston Apteekin kanta-asiakkaille tutkimusuutiskirjeenä ja Suomen Syöpäpotilaat ry tiedotti tutkimuksesta sähköisessä viestinnässään. Tutkimuksen kohderyhmänä olivat aikuiset, joilla oli lääkärin toteama syöpäsairaus ja jotka olivat käyttäneet syöpäsairautensa hoitoon lääkärin määräämä lääkehoitoa kyselyä edeltävän 12 kuukauden aikana. Tutkimusaineisto koostui 294 kohderyhmän potilaasta. Ensisijaisella tulosmuuttujalla (summamuuttuja) tutkittiin syöpäpotilaiden näkemyksiä lääkärin toteuttamasta biologisten lääkkeiden lääkevaihdosta. Vastaajilla oli keskimäärin myönteinen näkemys lääkärin toteuttamasta biologisten lääkkeiden lääkevaihdosta, mutta lääkevaihdon toteuttamiseen liittyi potilaiden kokemaa epävarmuutta (summamuuttuja: Cronbachin alfa 0,808; keskiarvo 3,29/5,00, 95 % lv 3,20–3,38). Myönteinen näkemys oli yhteydessä potilaiden myönteisiin näkemyksiin biosimilaarien ja geneeristen lääkkeiden ominaisuuksista, kuten tehosta, haittavaikutuksista ja käytettävyydestä sekä vastaajan korkeampaan koulutustasoon ja vähäisempiin huoliin yleisesti omasta lääkehoidostaan. Enemmistö (72 %) vastaajista tunnisti biologisen lääkkeen käsitteen, biosimilaari-käsite tunnistettiin heikommin (19 %). Vastaajien käytetyimmät lääketiedon lähteet syöpälääkityksestään olivat terveydenhuollon ammattilaiset (48–88 %) ja pakkausseloste (80 %). Syöpäpotilaiden näkemyksiin biologisten lääkkeiden lääkärin toteuttamasta lääkevaihdosta vaikuttivat useat tekijät, kuten näkemykset biosimilaarien ja geneeristen lääkkeiden ominaisuuksista. Vaikka useat syöpäpotilaat suhtautuivat keskimäärin myönteisesti lääkärin toteuttamaan biologisten lääkkeiden lääkevaihtoon, siihen liittyi usein epävarmuutta. Tulevaisuudessa tulisi tutkia tarkemmin syöpäpotilaiden tietämystä biologisista lääkkeistä ja biosimilaareista. Lisäksi tulisi tutkia millaista lääkeinformaatiota potilaat tarvitsevat päätöksenteon tueksi biologisten lääkkeiden lääkevaihdossa.
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(2017)Interindividual variability in drug responses can complicate the determination of drug doses and increase drug-related risks. The variability can be caused by pharmacokinetics or pharmacodynamics of drug. One significant factor giving rise to the variability in the pharmacokinetics is the genetic polymorphism of cytochrome P450 (CYP) enzymes. CYP2C19 and CYP2D6 are highly polymorphic enzymes and many of their polymorphisms are well-known. For both genes there exist null alleles producing the enzyme with complete lack of function and alleles producing increased enzyme activity. Additionally there are alleles of CYP2D6 leading to partially deficient enzyme function. Based on the genotype of the CYP gene individuals can be divided into four phenotype groups describing the enzyme activity: poor, intermediate, extensive and ultrarapid metabolizers. According to the clinical observations the pharmacokinetics of CYP2C19 and CYP2D6 substrates in the individuals genotyped as poor metabolizers often significantly differentiates from the pharmacokinetics in the individuals belonging to other phenotype groups. Between the other phenotype groups the pharmacokinetic variability caused by the genotype seems to be often covered by other reasons causing variability in the pharmacokinetics. The pharmaceutical industry could benefit from methods that could predict the interindividual variability in the drug responses before the clinical studies. The pharmacokinetic variability caused by the genetic polymorphism of CYP enzymes has been predicted with different kinds of static and dynamic physiologically based pharmacokinetic simulation models. The models have taken the CYP genotype into account by non-substratespesific or substratespesific methods. The models have succeeded to predict the clinically observed interindividual variability in the pharmacokinetics of substrates. The goal of this study was to find out if in vitro metabolism data obtained with human liver microsomes genotyped for CYP2C19 or CYP2D6 could be used to predict the interindividual variability in the pharmacokinetics of drugs. The effect of polymorphism on metabolism was examined by incubating the substrates with microsomes with different CYP2C19 or CYP2D6 genotypes. S-mephenytoin, omeprazole and Y1 (compound developed by the pharmaceutical company Orion Oyj) were used as substrates for CYP2C19. Neither the rate of metabolism of S-mephenytoin nor omeprazole appeared to be dependent on the CYP2C19 genotype, with the exception of the poor metabolizer genotype. Use of microsomes genotyped for the other CYP2C19 phenotypes to obtain predictive in vitro metabolism data might therefore not be reasonable. More significant dependence of the Y1 metabolism on the CYP2C19 genotype could not be completely excluded. When examining the effect of polymorphism on non-selective metabolic reactions, the activity of metabolizing enzymes other than the polymorphic enzyme should always be taken into consideration: in this study, CYP3A4 activity biased the results initially achieved with omeprazole and Y1. Dextromethorphan and bufuralol were used as substrates for CYP2D6 and their rates of metabolism correlated well with the CYP2D6 genotype. So microsomes genotyped for CYP2D6 could possibly be used to obtain predictive in vitro metabolism data. If genotyped microsomes are to be used in the pharmaceutical industry to predict the interindividual variability in the pharmacokinetics, factors increasing reliability of the results should be considered first and more studies should be conducted.
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(2023)Inhibition of the cytochrome P450 enzymes is one of the most significant factors causing drug-drug-interactions, and thus one of the most important objects of study at preclinical drug development. CYP-inhibition can be either reversible or irreversible. Although different inhibition mechanisms are well known, their evaluation in vitro is still challenging. Thus, the development of more accurate and efficient in vitro methods is important and as a continuous target of interest. Immobilized enzyme microreactors (IMER) have presumably several advantages over traditional in vitro methods and have been presented as a promising tool for drug metabolism studies in vitro. The purpose of this work was to evaluate the suitability of a novel flow-through based immobilized enzyme microreactor in determining the CYP enzyme kinetic parameters. The developed immobilization protocol is based on attaching biotinylated human liver microsomes to a thiolene-based microreactor coated with Streptavidin. To validate the developed method, the activity of the CYP2C9 enzyme was assessed using the recommended model reaction by authorities, that is 4-hydroxylation of diclofenac. The enzyme kinetic parameters i.e., enzyme affinity (Km) and activity (Vmax), determined with the developed IMER were comparable to the values previously published in the literature and determined in static in vitro conditions. In addition, the inhibition of CYP2C9 enzyme by four model inhibitors (fluconazole, nicardipine, sulfaphenazole and miconazole), was examined by determining the IC50 (half-maximal inhibitory constant) values for each compound and by monitoring the reversibility of the CYP2C9 enzyme for 90 minutes after the inhibitor was removed from the feed solution. The IC50 values determined with the developed method for all inhibitors were well in line with previous publications, showing fluconazole (IC50 22 µM) to be the weakest inhibitor of CYP2C9 enzyme and the other examined inhibitors caused more potent inhibition (IC50 for sulfaphenazole 1.3 µM; IC50 for miconazole 1.3 µM; IC50 for nicardipine 0.67-1.1 µM). The reversibility of the CYP2C9 enzyme was examined by removing the inhibitor from the feed solution and monitoring the recovery of the enzyme activity via diclofenac 4-hydroxylation. Based on the results obtained with developed IMER, the inhibition of fluconazole and sulfaphenazole was reversible and thus well in line with previous studies. In contrast, on account of data obtained with IMER, inhibition by miconazole and nicardipine was not reversible, although these compounds have previously been reported to be reversible CYP2C9 inhibitors in vitro, which may be due to the strong aggregation tendency of these compounds. The study shows that the developed flow-through based IMER is well suited for studying inhibition of CYP enzymes However, to utilize the developed technology in CYP enzyme inhibition research, it’s applicability in determining enzyme inhibition should still be evaluated with more comprehensively with several CYP isoenzymes.
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(2023)Lääkkeiden saatavuushäiriöt ovat yleistyneet maailmalla ja Suomessa viimeisten 20 vuoden aikana lisäten lääkealan toimijoiden työmäärää sekä kustannuksia. Saatavuushäiriöt aiheuttavat myös vaikeuksia potilaiden hoidon toteuttamiseen, tietyissä tilanteissa hoito saattaa joutua katkolle tai joudutaan käyttämään vaihtoehtoista lääkitystä, mikä saattaa heikentää potilaan saaman hoidon laatua. Tämän tutkimuksen tavoitteena oli tutkia kirjallisuuden perusteella syitä saatavuushäiriöiden taustalla. Toissijaisena tavoitteena oli löytää mahdollisesti toimintamalleja, mitkä mahdollistaisivat saatavuushäiriöiden määrän vähenemisen tai vaihtoehtoisesti vähentäisivät niiden vaikutuksia potilaiden hoitoon. Tutkimus tehtiin systemoituna kirjallisuuskatsauksena. Tutkimukseen valikoitui mukaan yhteensä kaikkiaan 24 sisäänottokriteerit täyttävää artikkelia. Kirjallisuushaku suoritettiin 20.10.2020 Sopusi ja Pubmed -tietokannoista. Kirjallisuushaku rajattiin koskemaan ajanjaksoa 2000–2020. Tietokantojen lisäksi kirjallisuutta haettiin manuaalisesti löydettyjen artikkeleiden lähdeluetteloista. Kirjallisuushaulla löydettiin yhteensä 2334 artikkelia ja duplikaattien poiston jälkeen jäljelle jäi 1783 artikkelia. Ensin artikkelit käytiin läpi otsikon sekä abstraktin perusteella ja viimeisessä vaiheessa koko tekstin perusteella. PRISMA-listaa käytettiin soveltuvin osin apuna katsauksen laatimisessa. Tähän systemoituun kirjallisuuskatsaukseen mukaan valituista julkaisuista 10 oli alkuperäisiäjulkaisuja artikkeleita ja 14 katsausartikkeleita. Alkuperäisistä tutkimusartikkeleista suurin osa (n=7) oli kvantitatiivisia tutkimuksia ja näistä neljä käsitteli saatavuushäiriöitä Yhdysvalloissa. Kvantitatiiviset tutkimukset olivat kaikki viranomaisten ylläpitämistä rekistereistä tehtyjä tutkimuksia. Laadullisia tutkimuksia alkuperäisissä artikkeleissa oli kolme, nämä olivat toteutettu puolistrukturoituina haastatteluina tai avoimina kyselyinä lääkealan toimijoille Systemoidun kirjallisuuskatsauksen tulosten perusteella saatavuushäiriöiden syyt voivat vaihdella eri markkinoiden välillä. Saatavuushäiriöiden taustalla olevien syiden teemat kuitenkin vaikuttivat tutkimuksen perusteella olevan hyvin samankaltaisia riippumatta markkinoista. Yleisimpinä syinä saatavuushäiriölle olivat tuotantoon liittyvät ongelmat, regulatoriset toimet sekä ekonomiset ratkaisut. Tulosten perusteella ensisijaisena ratkaisuna saatavuushäiriöiden vaikutusten minimointiin olisivat lääkkeiden valmistajien tarpeeksi ajoissa antamat ilmoitukset alkavasta saatavuushäiriöistä.
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(2010)Automated dose dispensing is an increasing field in which medicines are packaged mechanically into small one-dose pouches in portions of two weeks. Suitability of tablets for automated dose dispensing has not been researched systematically earlier. The study was made in collaboration with the dose dispensing unit of Espoonlahti pharmacy. The aim of the study was to define optimal characteristics for an automatically dispensed tablet from a viewpoint of the dose dispensing process to reduce breakings and transitions. Breaking means that tablet crumbles, splits up or breaks up otherwise during mechanical dose dispensing process. Transition means that tablet is dispensed in a wrong dose pouch. Percentually breakings and transitions occur very little, but quantitatively plenty and increasingly when automated dose dispensing is becoming more common. Breakings and transitions cause plenty extra work because of correcting pouches, so their amount should be aimed to reduce. In addition, the aim is to find out matters to enquire from the manufacturers of medicines that would help concluding whether a product is suitable for automated dose dispensing based on written information. Results of the study indicate that to reduce breakings and transitions, an optimal tablet product for dose dispensing is rather small or middle sized, coated, strong and without a breakline and the optimal relative humidity of air in the product room of dose dispensing unit would be around 30 - 40 %. Matters to enquire from the manufacturers of medicines besides size, coating, breaking strength and breakline are stability of the product outside of its original package and light, heat and moisture sensitiveness of the product. Besides breakings and transitions, also stability of a moisture sensitive acetylsalicylic acid product (Disperin 100 mg) was investigated in 25 °C/60 % RH because air humidity in the product room is not adjustable. Duration of the test was four weeks. It is enough since it is the maximum time that tablets are outside their original packages during drug dispensing process before use. Tablets were kept in opened original container (bottle), in closed original container, in cassette of dispensing machine and in two different dose pouches (new material and the one in use). According to the results, cassettes are protecting tablets from moisture as poorly as an opened bottle. Instead, new pouch material protects tablets better than the material in use. Results of Raman spectroscopy measurements indicate no change in acetylsalicylic acid to salicylic acid during four weeks test. Moisture affects to tablets by decreasing breaking strength, which may cause more breakings. Air humidity should be adjusted in product rooms or tablets should be unpacked into cassettes as near operating the machine as possible to prevent breakings. Especially when air humidity is high. Among others, a heat sensitive drug product was researched because of the seaming unit of dose dispensing machine which is radiating heat of about 75 °C to pouches if machine is pulled over in the middle of work. Study was performed with variable temperature XRPD. Results of the study of heat sensitiveness indicate that 75 °C for 60 minutes doesn't induce changes in carbamazepine tablet (Neurotol 200 mg). However, results of the study reveal that researched product did not contain the most heat sensitive form of carbamazepine, so other heat sensitive drug products should be examined to get more information about effects of heat.
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(2023)For pediatric patients, it is often necessary to resort to off-label use of available commercial products. This may require manual modification of the preparations, which may result in reduced dose accuracy. In the past, there have been discussions about the potential of 3D printing technologies for on-demand manufacturing of medicines in hospitals. Printing technologies can be used to tailor medicines to the individual needs of patients. This could be a possible solution to the lack of commercial products for pediatric patients, for example. Semi-solid extrusion is a printing technique that could potentially be used in the future in hospitals. This study aims to design the simplest possible excipient composition for a printing material for semi-solid extruded preparations for pediatric patients. The finished products will be examined to determine the type of products achieved with this printing method and excipient composition. In addition, the suitability of semi-solid extrusion in a hospital environment will be observed and evaluated. Printing was performed with a pneumatic bioprinter. The desired formulations could not be prepared with a printing material containing only a gel former (poloxamer 407) and a solvent (water). Therefore, a filler (microcrystalline cellulose) was added to the printing material to improve the mechanical strength of the preparations. The model drug used in the study was warfarin sodium and the target strengths of the preparations were 0.1 mg, 0.5 mg and 1.0 mg. The preparations were dried at room temperature for 22-23 hours. The tablets produced in the study were small (diameter less than 7 mm, height less than 2 mm) grid-structured preparations. The method was successful in producing tablets of uniform mass. For all strengths, the tablets passed the European Pharmacopoeia test for uniformity of content of single-dose preparations. Only the 0.5 mg strengths passed the test of uniformity of dosage units. The excipient composition should still be optimized to improve the mechanical strength of the products. The overall preparation time of the formulations should be reduced, for example by shortening the drying time, to make semi-solid extrusion suitable for extemporaneus preparations in hospitals.
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(2012)3D-imaging is based on combining two or more pictures to form one three-dimensional picture. Most of the methods used provide only surface pictures, but tomography acquires also information about the inside-structure of investigated material. Young's modulus is a method, which has been used for long time to determine toughness hard materials, such as steal. In traditional method a beam-shaped piece is bent. When the size of piece, used force and amount of bending are known, Young's modulus of piece can be calculated. Although the method has traditionally been used to research very hard materials, it has been applied without changes with pharmaceutical materials. It is, however, open to the question whether or not the method is appropriate for those materials. There are also methods to determine Young's modulus based on compressing a tablet or using ultrasound. Determining tablet's toughness with ultimate strength test is complicated because it breaks tablet. For that reason it would be good to find compensatory methods to measure strength of tablet. The aim of the study was to validate Flash Sizer 3D appliance, which is used in 3D-imiging. Another goal was to investigate possible correlations between 3D-imiging, Young's modulus and traditional ultimate strength method. Lastly, the feasibility of Young's modulus as a substitute for traditional ultimate strength measurement in self life studies was investigated. Flash Sizer 3D was validated by measuring particle size distribution of pellets, which were made of microcrystalline cellulose (Cellets). Sizes of the investigated pellets were 100 µm, 200 µm and 500 µm. Also binary mixture of 100 µm and 200 µm was investigated. From microcrystalline cellulose was made tablets and 3D-pictures were taken. Ultimate strength test was made for half of the tablets. Young's modulus was measured from half of the tablets in tableting day, day after that and nine days after tableting. Results show that Flash Sizer 3D is suitable for investigating bigger Cellet. With smaller particles distinguishing of tablets wasn't probably good enough. Still it seems to be quite good method to determine surface roughness of tablet. Young's modulus seems to be very promising as compensating method for traditional ultimate strength measurement. In future in self life studies tablets hardness might be able to investigate by measuring Young's modulus and not measuring ultimate strength. If correlation between Young's modulus and solubility meets the case, Young's modulus might also replace also solubility measurements in self life studies.
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(2024)Tablets are solid medicinal products that are produced by compressing tablet mass in a tablet press. The reproducible and reliable functionality of the manufacturing process, facilities and equipment used in the manufacturing process of medicinal products must be validated. There must be a marketing authorisation for the sale of medicinal products, and pharmaceutical companies must comply with current legislation, Good Manufacturing Practices, and other binding guidelines. After marketing authorisation, certain changes, such as significant changes related to the manufacturing process or raw materials, must be notified to the competent authority by means of a variation application. Depending on the extent of the change, a revalidation and/or a regulatory approval before implementing the change may be required, which makes manufacturing process modification slow, expensive, and laborious. However, the pressure to enhance pharmaceutical manufacturing processes and reduce costs is prevailing. In this study, Lean Six Sigma methods were applied to enhance the manufacturing process of a tablet product. The study was divided into two parts. In the first part of the study, the objective was to investigate the manufacturing process of a tablet product X and to identify improvement actions that would make the manufacturing process of the tablet product X more efficient by achieving material and time savings. In addition, improvement actions were prioritized based on the benefits achieved by the improvement actions and the difficulty of implementation. The second part of the study was based on the improvement action selected from the first part of the study. The objective of the second part of the study was to investigate the factors influencing the amount of mass loss during tableting and how the amount of mass loss can be influenced. In this study, mass loss during tableting referred to the dusty mass inside the tablet press during tableting, which is removed from the tablet press to the equipment dedusting system. The purpose of the practical experiment conducted in the tablet production, which was part of the second part of the study, was to investigate how the magnitude of the exhaust airflow and fan power of the equipment dedusting system affects the amount of mass loss generated during tableting. Based on the experiment, adjusting the power of the equipment dedusting system can affect the amount of mass loss generated during tableting. However, further studies are needed to ensure that no dusty mass remains in the piping of the dedusting system or in tablet press at low exhaust airflow and fan power values during tableting. If the results are applied to other tablet products, the effect of a different formulation on powder dusting should be considered. Adequate dedusting during tableting is important so that the dusty mass inside the tablet press is removed as intended, facilitating the cleaning of the tablet press after tableting and reducing the risk of cross-contamination and exposure of workers to dust. The conclusion of this study is that Lean Six Sigma methods can be used more extensively to enhance the manufacturing processes of both tablet products and other pharmaceutical dosage forms and to reduce loss generated during the manufacturing process without revalidation or changes to a valid marketing authorisation.
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(2010)It was given a task to the Finnish Medicines Agency (Fimea) to prepare a national Medicines Information Strategy. The strategy process can be divided into four stages: 1) the collection and analysis of the information, 2) the determination of the strategy and the vision, 3) the realization and 4) the follow-up stage of the strategy. In the European Union (EU) the High Level Pharmaceutical Forum has drawn up the criteria for the high quality medicines information (MI) and the recommendations to improve the quality and availability of the MI directed to the consumers. The most significant medicines related political actions in Finland in the 21st century are the Medicine policy 2010 -document, the strategies of the National Agency for Medicines and the TIPPA-project. The objective of the Master's thesis was to produce the information to Fimea's MI work. The electric questionnaire was drawn up in the Ministry of Social Affairs and Health. After a pilot test, the questionnaire was sent by email to all national Medicines Authorities in EU (n=27) in November 2009. The purpose of the questionnaire was to find out 1) the significance of the medicine information in the national legislation, 2) the possible MI strategies and 3) the control mechanisms of the medicine information directed to the consumers. The medicines information strategies were found in the United Kingdom (UK), Italy and Germany. Furthermore, the strategy process was unfinished in four countries. In the strategy of the UK 25 concrete actions were presented during a three-year strategy period to improve the quality and availability of the MI and to improve the cooperation between public and private actors. The information and communication technology (ICT) was in the centre of the medicines information offered to the consumers. ICT was utilised by publishing Patient Information Leaflets in Internet and by developing medicines information web pages, digitally patient counseling services and quality certificates. The results of the survey can be utilised as a part of the Fimea's Medicines Information Strategy process. Further studies, for example an analysis of the interest groups, are needed before the preparation of the national strategy. Furthermore, experiences of the implementation of the strategy and the results reached in the UK should be clarified.
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