High-throughput assay to simultaneously evaluate activation of CYP3A and the direct and time-dependent inhibition of CYP3A, CYP2C9, and CYP2D6 using liquid chromatography-tandem mass spectrometry

Abstract

1. In the early stages of drug discovery, adequate evaluation of the potential drug–drug interactions (DDIs) of drug candidates is important. Several CYP3A activators are known to lead to underestimation of DDIs. These compounds affect midazolam 1'-hydroxylation but not midazolam 4-hydroxylation.

2. We used both metabolic reactions of midazolam to evaluate the activation and inhibition of CYP3A activators simultaneously. For our CYP inhibition assay using cocktail probe substrates, simultaneous liquid chromatography-tandem mass spectrometry monitoring of 1'-hydroxymidazolam and 4-hydroxymidazolam for CYP3A was established in addition to monitoring of 4-hydroxydiclofenac and 1'-hydroxybufuralol for CYP2C9 and CYP2D6.

3. The results of our cocktail inhibition assay were well correlated with those of a single inhibition assay, as were the estimated inhibition parameters for typical CYP3A inhibitors. In our assay, a proprietary compound that activated midazolam 1'-hydroxylation and tended to inhibit 4-hydroxylation was evaluated along with known CYP3A activators. All compounds were well characterised by comparison of the results of midazolam 1'- and 4-hydroxylation.

4. In conclusion, our CYP cocktail inhibition assay can detect CYP3A activation and assess the direct and time-dependent inhibition potentials for CYP3A, CYP2C9, and CYP2D6. This method is expected to be very efficient in the early stages of drug discovery.

Keywords:

• Allosteric activation
• midazolam 4-hydroxylation
• cytochrome P450
• CYP3A
• cocktail
• CYP inhibition
• DDI
• LC-MS/MS

Acknowledgements

The authors thank all members of Kissei pharmaceutical Co., Ltd. who assisted with this study.

Disclosure statement

The authors are employees of Kissei Pharmaceutical Co., Ltd.

Additional information

Funding

Funding for this research was provided by Kissei Pharmaceutical Co., Ltd.