Browsing by Subject "entsyymi-immobilisointi"
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(2017)Cytochrome P450 (CYP) enzymes are important catalysers in the first phase of drug metabolism. Roughly two thirds of drugs are oxidized via CYP enzymes, which enable the further modification of drugs, and their excretion. In this thesis, human liver microsomes containing the main hepatic CYP enzymes were immobilized on thiol-ene based micropillar arrays and their stability was evaluated using a CYP2C9 isoenzyme specific luminescent substrate, Luciferin-H. The aim of the study was to develop microfluidic immobilized enzyme reactors (IMERs) for studying enzyme kinetics and drug-drug interactions. For this purpose, the instability issues associated with previously reported CYP-IMERs were carefully addressed. The CYP immobilization protocol used was based on a protocol previously developed in the context of other research projects and relied on biotinylation of human liver microsomes (HLM) with help of fusogenic liposomes. The biotinylated HLMs were then attached to the streptavidin-modified thiol-ene surfaces. The CYP activity was determined by utilizing microfluidics under continuous flow conditions (typically 5 μL/min) in the presence of NADPH. The luminescent metabolite formed by the CYP2C9 enzymes was quantified with a commercial well-plate reader from fractions collected at the microreactor outlet. Half-life was used to compare the differences between enzyme stabilities reached via different immobilization conditions. The effects of flow rate and reaction temperature on the stability of the CYP-IMERs was evaluated together with addition of antioxidative agents and reactive oxygen species (ROS) scavengers. Different functionalization steps as well as storage time and conditions were studied. With Luciferin-H as the model substrate of CYP2C9, the CYP-IMERs showed higher activity and stability at room temperature than at +37 °C. The peak activity could be increased via optimization of the immobilization protocol, though long-term storage diminished the peak activity. The activity of the IMERs typically attenuated within 1-2 hours with little or no improvement achieved via optimization of the immobilization or operation conditions. Only upon addition of the ROS scavengers, the peak activity and stability of the CYP-IMERs could be slightly improved. After functionalization, the IMERs maintained their activity until the time of use when stored in +4 °C for up to 2 weeks, but re-use of IMERs was not possible.
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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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