Rapidly distinguishing reversible and irreversible CYP450 inhibitors by using fluorometric kinetic analyses

Abstract

In this study we have evaluated the reliability of a fluorescence-based method used for rapid identification of irreversible CYP inhibitors (mechanism-based inhibitors). This was accomplished by comparing the time-dependence pattern of IC50 values from fluorometric kinetic measurements. For irreversible CYP inhibitors, IC50 values decreased as incubation proceeded. This was due to progressive inactivation of corresponding enzymes by reactive metabolites generated during the incubation. This change pattern was confirmed using a number of known irreversible CYP inhibitors, including furafylline, midazolam, erythromycin, clarithromycin, oleandomycin, 17alpha-ethynylestradiol and verapamil. The pattern was different in reversible inhibition, depending upon the compounds tested in the fluorometric kinetic assay. For some compounds, such as clotrimazole, IC50 values remained relatively stable, whereas other compounds, such as miconazole, terfenadine and ketoconazole showed a significant increase with incubation time. Monitoring tested compounds by LC-MS/MS during the incubation confirmed that increases of IC50 were probably caused by the loss of inhibitors, resulting from either metabolic degradation, or non-specific binding to microsomal proteins.

Mechanism-Based Inhibition: Deriving KI and kinact Directly from Time-Dependent IC50 Values

“…As reported by Yan et al, 2002 andNaritomi et al, 2004, irreversible CYP inhibition can easily be stopping the enzymatic reactions. Therefore, estimating IC50 values over time using microtiter plate assays is a simple and attractive method to detect irreversible inhibitors (Naritomi et al, 2004).…”

“…This parameter is related to the time-dependent decrease of IC50 values for irreversible inhibition (Yan et al, 2002). In this situation, the Cheng-Prusoff equation results in erroneous predictions.…”

Stress?restress evokes sustained iNOS activity and altered GABA levels and NMDA receptors in rat hippocampus

“…KCZ, which is a well-established pharmacological tool used to inhibit adrenal steroid synthesis, binds to the heme iron of cytochrome 450P (CYP450) isoforms mediating steroid hydroxylation reactions (Wolff et al 1993;Yan et al 2002). Thus, early increases in glucocorticoids immediately after the stressor may be critical for the protracted effects on NOS.…”

Prediction of Drug-Drug Interactions based on Time-Dependent Inhibition from High Throughput Screening of Cytochrome P450 3A4 Inhibition

“…A screening method for CYP inhibition using fluorescent substrates has been reported. 11,12) This method could be utilized to find potential mechanism-based inhibition since mechanismbased inhibitors enhance the degree of inhibition according to the preincubation time, so-called time-dependent inhibition (TDI). This method is suitable for use with HTS because it enables a rapid measurement of many compounds.…”