Validation of cytochrome P450 time-dependent inhibition assays: a two-time point IC₅₀ shift approach facilitates k_inact assay design

Abstract

1. Recent guidance from the US Food and Drug Administration (USFDA) has advocated testing of time-dependent inhibition of cytochrome P450 (CYP), which can be addressed by performing IC₅₀ shift as well as K_I/k_inact determinations.

2. Direct (IC₅₀, K_i) and time-dependent inhibition (IC₅₀ shift, K_I/k_inact) assays were validated in human liver microsomes with liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis for the following enzyme/substrate/inhibitor combinations: CYP1A2/phenacetin/alpha-naphthoflavone/furafylline, CYP2C8/amodiaquine/montelukast/gemfibrozil-1-O-β-glucuronide, CYP2C9/diclofenac/sulfaphenazole/tienilic acid, CYP2C19/S-mephenytoin/S-benzylnirvanol/S-fluoxetine, CYP2D6/dextromethorphan/quinidine/paroxetine, and CYP3A4/midazolam/testosterone/ketoconazole/azamulin/verapamil/diltiazem. IC₅₀ shift assays were performed with two pre-incubation time points (10 and 30 min) to facilitate k_inact assay design.

3. Data obtained show good agreement with literature values. For rapid acting inhibitors, such as azamulin/CYP3A4 and tienilic acid/CYP2C9, the IC₅₀ shifts were similar at both time points suggesting a short maximum pre-incubation time with closely spaced time points for the K_I/k_inact assay. Slow acting inhibitors (such as verapamil/CYP3A4 or S-fluoxetine/CYP2C19) showed an increase in IC₅₀ shift between 10 and 30 min suggesting a longer maximum pre-incubation time with wider spacing of time points for K_I/k_inact.

4. The two-time point IC₅₀ shift experiment proved to be an excellent method for the selection of appropriate K_I/k_inact assay parameters and is suitable for the routine analysis of P450 inhibition by drug candidates.

Keywords::

• Cytochrome P450
• time-dependent inhibition
• inhibition
• human liver microsomes (HLM)
• k^_inact
• IC₅₀ shift

Acknowledgements

Declaration of interest: All authors are current or past employees and share holders of BD (Becton, Dickinson & Company), which supplies some of the reagents used in this work.