HLM chip – A microfluidic approach to study the mechanistic basis of cytochrome P450 inhibition using immobilized human liver microsomes
•Microfabricated flow-through assays enable the study of time-dependent drug responses.•Microfluidic HLM chips have enzyme kinetics comparable to static enzyme incubations.•Microfluidic HLM chips enable facile identification of irreversible enzyme inhibitors.•HLM chip assays suggest that miconazole...
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Veröffentlicht in: | European journal of pharmaceutical sciences 2024-06, Vol.197, p.106773-106773, Article 106773 |
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Zusammenfassung: | •Microfabricated flow-through assays enable the study of time-dependent drug responses.•Microfluidic HLM chips have enzyme kinetics comparable to static enzyme incubations.•Microfluidic HLM chips enable facile identification of irreversible enzyme inhibitors.•HLM chip assays suggest that miconazole has prolonged inhibitory impact on CYPs.
Cytochrome P450 (CYP) system is a critical elimination route to most pharmaceuticals in human, but also prone to drug-drug interactions arising from the fact that concomitantly administered pharmaceuticals inhibit one another's CYP metabolism. The most severe form of CYP interactions is irreversible inhibition, which results in permanent inactivation of the critical CYP pathway and is only restored by de novo synthesis of new functional enzymes. In this study, we conceptualize a microfluidic approach to mechanistic CYP inhibition studies using human liver microsomes (HLMs) immobilized onto the walls of a polymer micropillar array. We evaluated the feasibility of these HLM chips for CYP inhibition studies by establishing the stability and the enzyme kinetics for a CYP2C9 model reaction under microfluidic flow and determining the half-maximal inhibitory concentrations (IC50) of three human CYP2C9 inhibitors (sulfaphenazole, tienilic acid, miconazole), including evaluation of their inhibition mechanisms and nonspecific microsomal binding on chip. Overall, the enzyme kinetics of CYP2C9 metabolism on the HLM chip (KM = 127 ± 55 µM) was shown to be similar to that of static HLM incubations (KM = 114 ± 14 µM) and the IC50 values toward CYP2C9 derived from the microfluidic assays (sulfaphenazole 0.38 ± 0.09 µM, tienilic acid 3.4 ± 0.6 µM, miconazole 0.54 ± 0.09 µM) correlated well with those determined using current standard IC50 shift assays. Most importantly, the HLM chip could distinguish between reversible (sulfaphenazole) and irreversible (tienilic acid) enzyme inhibitors in a single, automated experiment, indicating the great potential of the HLM chip to simplify current workflows used in mechanistic CYP inhibition studies. Furthermore, the results suggest that the HLM chip can also identify irreversible enzyme inhibitors, which are not necessarily resulting in a time-dependent inhibition (like suicide inhibitors), but whose inhibition mechanism is based on other kind of covalent or irreversible interaction with the CYP system. With our HLM chip approach, we could identify miconazole as such a compound that nonselectively i |
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ISSN: | 0928-0987 1879-0720 |
DOI: | 10.1016/j.ejps.2024.106773 |