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Browsing by Subject "validointi"

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  • Kallio, Arttu (2014)
    Cytochrome P450 (CYP) -enzymes are one of the most important enzymes in the metabolism of xenobiotics. Because many xenobiotics are metabolized with each other by the same CYP-enzymes, it is possible that metabolic interactions will take place. These interactions can be the inhibition or induction of the metabolism of another xenobiotic. The interaction can be harmful e.g. when it causes an accumulation of a toxic metabolite or when it inhibits the metabolism of an active drug substance. The aim of this study was to develop a quantitative method for determining metabolic interactions between drugs and environmental chemicals in human liver microsome (HLM) incubations. HLMs contain high concentrations of CYP-enzymes, enabling the use of CYP-model reactions for observing interactions. The model reactions chosen for this study were O-deethylation of phenacetin (CYP1A2), 7-hydroxylation of coumarin (CYP2A6), 4'-hydroxylation of diclofenac (CYP2C9), 1'-hydroxylation of bufuralol (CYP2D6) and 6β-hydroxylation of testosterone (CYP3A4). Michaelis-Menten constants (Km) and maximal enzymatic activities (Vmax) were determined for each model reaction. The suitability of the model reactions for inhibition studies was assessed with specific inhibitors. The quantitative method was developed for an ultra-high performance liquid chormatograph (UPLC) and for a quadrupole time of flight mass spectrometer (QTOF). Samples were ionized with electrospray ionization (ESI) using positive mode. Device parameters were the same for all the metabolites. The analytical method validation was partly performed according to ICH (International Conference on Harmonisation) guidelines. A sufficient linearity (R2>0,99) and specificity was achieved for the quantitative method. The achieved limits of quantitation (LOQ) were low enough (1-120 nM) for quantitation of the small concentrations of the metabolites formed in the inhibition assays. The measurement reproducibility and the reproducibility and accuracy of the method did not fulfill the acceptance criteria for all the metabolites. Improvement of the results should be tried by e.g. exploring different device parameters. 1'-hydroxydiclofenac was found likely to degrade in the matrix solution because of the acidic conditions, making the reliability of the results poor for this metabolite. The Km value obtained for coumarin differed markedly from literature values, which can be due to a too long incubation time. Therefore, incubation conditions should be optimized for this model reaction in coming studies. The Km values obtained for the model reactions of CYP1A2, CYP2D6 and CYP3A4 were similar to those found in literature. Also the IC50 values were quite well within the range of values reported in literature for the inhibitors of the above mentioned model reactions. The effects of four different polymers, F68, F127, Tetronic 1307 and polyvinyl alcohol (PVA) on the enzyme activities were also studied, at a concentration of 1 mg/ml. In principal, at this concentration the polymers did not cause significant changes in the enzyme activities, although inhibition of the CYP2C9 could have been significant. However, the reliability of CYP2C9 model reaction was found to be poor with the used method. In the future this developed method should be further validated, and the incubation conditions for the model reaction of CYP2A6 should be optimized. After this, the IC50 values for the polymers could be studied to get more reliable information about their potential CYP-inhibition properties.
  • Kilpiö, Tommi (2021)
    Plant cell culture can be used for the production of valuable secondary metabolites. Inspired by the previous studies focusing on capsaicinoid production, this study aimed for establishing plant cell cultures of Capsicum chinense to produce capsinoids. Capsinoids are non-pungent capsaicinoid analogues with potential health benefits. Another aim of this study was to determine the α-solanine content in Capsicum plants and cell cultures to ensure that no toxic amounts are formed during the cell culture. Cell cultures of non-pungent Capsicum chinense cultivars, Trinidad Pimento and Aji Dulce strain 2, were established, and the cultures were fed with intermediates, vanillin and vanillyl alcohol, to enhance the production. In addition, cell cultures of extremely pungent Trinidad Scorpion cultivar were established and they were fed with vanillyl alcohol to study if this would result in formation of capsinoids instead of capsaicinoids. A high-performance liquid chromatography (HPLC) method with UV detection was validated for determining the capsiate contents of the cell culture samples and fruit samples for comparison. To analyze the α-solanine content of the cell culture samples and leaves and flowers of three cultivars belonging to three different Capsicum species, an HPLC-UV method was validated for this purpose as well. Despite validating a sensitive and specific method for capsiate analysis, no detectable amounts of capsiate were detected in any of the cell culture samples. Cell cultures of pungent cultivars did not produce detectable amounts of capsaicinoids either. Results from analyzing the real fruit samples were in accordance with previous literature reports, and Aji Dulce fruits were found to contain higher amounts of capsiate compared to Trinidad Pimento, although having only one indoor grown Aji Dulce fruit analyzed limits the reliability. The analytical method for determining α-solanine content had problems with internal standard and specificity. This method could be used for making rough estimates about the possible α-solanine content. No hazardous amounts were detected in any of the cell culture samples. Only one sample consisting of Aji Dulce young leaves could contain α-solanine slightly above the limits set for commercial potatoes. Results with flowers of Rocoto San Pedro Orange (C. pubescens) and Aji Omnicolor (C. baccatum) were inconclusive and it couldn’t be ruled out that they might contain large amounts of α-solanine. The reason why capsinoids, or even capsaicinoids, were not detected in the cell culture samples remains unsolved, but it could be speculated that capsinoids might degrade in the cell culture environment or that selection of cultivar or cell line is critical. This study gave further proof to the previous assumptions that chili leaves are safe and should not contain notable amounts of α-solanine.