Browsing by Subject "immobilized enzyme microreactors"

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.