Browsing by Subject "drug metabolism"
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(2021)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.
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(2021)Drug metabolism is a series of enzyme catalysed processes that modify foreign compounds into a form that is more easily excreted from the body. Compounds can affect the activity of metabolizing enzymes and this may lead to toxic concentrations of a drug that is metabolized via the enzyme. With prodrugs, on the other hand, the drug might not achieve its biologically active form and therefore the treatment will not be effective. Recognizing and preventing metabolic interactions is important already in the early stages of drug discovery and development. Cytochrome P450 (CYP) enzyme inhibition is one of the major reasons for adverse drug-drug interactions (DDIs). The inhibition can be time-dependent (TDI), which means that the potency of inhibition increases over time. TDI may be reversible or irreversible, latter being more severe as new enzymes need to be produced in the body to restore the enzymatic activity. IC50 shift assay is a method that gives information of new compounds potential to cause TDI. IC50 shift assay does not show whether the TDI is reversible or irreversible, however further studies, e.g. dialysis assay, can be conducted to find it out. If the study compound is irreversibly bound to the enzyme, the enzyme activity should not recover in the dialysis. The aim of this master’s thesis was to develop a dialysis method that could determine the reversibility of the TDI observed in the IC50 shift assay. A dialysis method conducted with microsomes is described in earlier literature. Known inhibitors (both time-dependent and direct) for four CYP isoforms were studied in this work: CYP1A2 (furafylline and fluvoxamine), CYP2C9 (tienilic acid and sulphaphenazole), CYP2D6 (paroxetine and quinidine) and CYP3A4 (verapamil, azamulin and ketoconazole). IC50 shift assays were conducted to each inhibitor before the dialysis experiment. The studied compounds behaved in the dialysis assay mostly as assumed based on the literature. The workflow from IC50 shift assay to dialysis assay worked successfully and the IC50 shift data could be utilized when choosing the test concentrations for dialysis assay. Both the IC50 shift assay and dialysis assay were reproducible and the deviations between replicates and separate studies were relatively low. The method still requires some optimizing, but so far, the results are promising. In the future the dialysis method may be part of in vitro CYP inhibition studies at Orion Pharma.
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(2020)Statins are a commonly used group of drugs that reduce the cholesterol levels in blood and have been shown to reduce cardiovascular morbidity and mortality. However, a considerable percentage of patients experience adverse effects during statin treatment. Statin adverse effects have been associated with genetic polymorphisms and drug-drug interactions that affect the elimination and active transport of these drugs. A more comprehensive knowledge of statin metabolism may be a step towards better management of statin treatments. Statin metabolism both in vivo and in vitro has been subject of study for years. In vitro incubation conditions may considerably affect the observed clearance, and results obtained with different methods or in different laboratories may not be directly comparable to each other. No single in vitro study on a wide panel of statins has previously been conducted. Six statins and some of their metabolites, fourteen compounds in total, were included in the study. The intrinsic clearance (CLint) of these molecules was investigated in vitro on human liver microsomes (HLM) and a panel of eleven cytochrome P450 (CYP) enzymes recombinantly expressed in E. coli. Observed CLint values for each compound in HLM and for each compound-CYP pair with observed depletion were calculated. The percentual contributions of each CYP enzyme to the metabolism of the compounds was calculated. The results obtained with recombinant CYP enzymes (rcCYP) were complemented with studies on HLM with specific chemical inhibitors of CYP enzymes. In this study the metabolism of statin lactones seemed to be faster than the metabolism of the corresponding statin acids. Atorvastatin lactone, 2-hydroxy atorvastatin lactone, 4-hydroxy atorvastatin lactone and simvastatin were extensively metabolized. Atorvastatin, 2-hydroxy atorvastatin, 3R,5S-fluvastatin, 3S,5R-fluvastatin, pitavastatin lactone and simvastatin acid showed intermediate metabolism. 4-hydroxy atorvastatin, pitavastatin, pravastatin and rosuvastatin rates of metabolism were below quantification limit. CYP3A4 had a major role in the metabolism of atorvastatin and its metabolites, simvastatin and simvastatin acid. CYP3A4 also had activity towards pitavastatin lactone. CYP2C9 had a high activity towards both 3R,5S-fluvastatin and 3S,5R-fluvastatin. CYP2D6 may play a part in the metabolism of pitavastatin lactone. CYP2C8 may have some activity towards simvastatin and simvastatin acid. The data is mostly in agreement with previous in vitro and in vivo studies regarding both the metabolism rate of statins and the contributions by different CYP enzymes to the metabolism of statins. Due to the screening nature of the study and some methodological constraints, these data should be considered as preliminary and require confirmation in further studies.
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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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