Browsing by Subject "lääkeainemetabolia"

Pharmaceutical contaminants in waste and surface waters have been recognized as an emerging risk to environmental health. Bioaccumulation of pharmaceuticals increases the risk of adverse effects in off-target species, as the chemical concentration within the organism exceeds the concentration of the surrounding environment. An organism’s ability to metabolize foreign organic compounds influences the likelihood of bioaccumulation. Current methods for predicting bioaccumulation in aquatic organisms are labour intensive or too simplistic to cover the variety of chemical and physiological processes involved and may lead to over or underestimations of environmental risk. A promising approach to improve bioaccumulation predictions, without the need of excessive animal testing, is to incorporate in vitro biotransformation data into computational models. The primary aim of this study was to assess whether selected pharmaceuticals (diclofenac, gemfibrozil, haloperidol, levomepromazine, levonorgestrel, sertraline and risperidone), that are well metabolized in humans through key biotransformation pathways, are metabolized by rainbow trout (Oncorhynchus mykiss) liver enzymes under physiologically relevant conditions (11°C, pH 7.8). A secondary aim was to produce fish in vitro intrinsic clearance (CLint, in vitro) data, that could potentially be used as input in computational models to predict bioaccumulation. In vitro biotransformation was studied using a single vial approach according to the Organisation for Economic Co-operation and Development (OECD) Test Guideline 319B: Determination of in vitro intrinsic clearance using rainbow trout liver S9 sub-cellular fraction (RT-S9). Depletion of the test compounds were measured during a 3-hour incubation period. High-performance liquid chromatography with ultraviolet detection (HPLC–UV) was used for qualitative and quantitative analysis of the samples. Levomepromazine, levonorgestrel and sertraline showed significant substrate depletion compared to negative controls while gemfibrozil, haloperidol, and risperidone did not seem to be metabolized. The results for verapamil were inconclusive. Levomepromazine displayed a higher in vitro intrinsic clearance rate (26 ml/h/g liver) than diclofenac (6.2 ml/h/g liver). These results are in accordance with previous studies and support the notion that a direct comparability between fish and human metabolism cannot be assumed, highlighting the need of fish in vitro biotransformation studies. The apparent lack of in vitro metabolism of risperidone, haloperidol, and gemfibrozil combined with their lipophilicity suggest that they are more likely to accumulate within rainbow trout, compared with the compounds that showed depletion during the assays, although repetitions and additional studies are needed to confirm this.

Cytochrome P450 (CYP) enzyme inhibition is one of the most common reasons for adverse drug-drug interactions. An especially harmful form of inhibition is time-dependent inhibition (TDI) in which the inhibition potency increases over time and persists even after discontinuation of the drug. Both direct and time-dependent inhibition can be efficiently screened with the so-called cocktail method containing several CYP-selective probe substrates in a single reaction mixture. This method is practical especially in ADME studies of drug development, as it offers lower costs, consumption of fewer reagents and faster implementation in comparison to conventional methods. In addition, the cocktail method can be used to establish new diagnostic CYP inhibitors in vitro. The aim of this Master’s thesis was to participate in the development and optimization of a new cocktail assay method. The method was developed for screening of major drug-metabolizing CYP enzymes in vitro both in a direct and time-dependent manner using pooled human liver microsomes. Based on preliminary testing, included probe substrates were divided into two cocktails to avoid significant inter-substrate interactions: cocktail I containing tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4, and cocktail II containing coumarin/CYP2A6, (S)-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2. First, cocktail incubation conditions were optimized, followed by the determination of probe reaction kinetics, kinetic parameters (Km, Vmax) and inter-substrate interactions with single- or dual-substrate incubations. Finally, suitable probe substrate concentrations and the composition of cocktails was evaluated based on the obtained results. As a result of assay optimization, optimal incubation conditions for yet unoptimized cocktail II were established. In optimized incubation conditions, all probe reactions exhibited saturable Michaelis-Menten kinetics except for tacrine 1-hydroxylation (CYP1A2), which exhibited biphasic kinetics instead (Km1: 7.36, Km2: 517). The selected probe substrate concentrations were all below or near their respective Km values except for (S)-mephenytoin 4’-hydroxylation (40 µM vs. Km of 12.5 µM); however, its concentration could not be reduced in order to maintain sufficient metabolite formation for UHPLC-MS/MS-analysis. Dual-substrate incubation assays demonstrated a need for the reduction of bupropion concentration below 100 µM due to its inhibitory effects on CYP2C8 and CYP3A4. In addition, chlorzoxazone/CYP2E1 and testosterone/CYP3A4 were tested as complementary probe substrates for the cocktails; however, they proved to be unsuitable for both cocktails due to significant interactions (>40% inhibition). Prior to the deployment of the method, some adjustments of probe substrate concentrations are still required in addition to consideration of the suitability of less commonly used CYP3A4 and CYP2E1 probe reactions to improve cocktail coverage. Lastly, validation of the method with known time-dependent model inhibitors should also be conducted. Besides to improvement of the cocktails, new information was generated on inter-cocktail probe-probe interactions and enzyme kinetics of probe reactions, especially for the less-studied astemizole O-demethylation (CYP2J2) and tacrine 1-hydroxylation (CYP1A2). Generated information can be used, for example, in the development of new cocktails.

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.