Cocktail-substrate assay system for mechanism-based inhibition of CYP2C9, CYP2D6, and CYP3A using human liver microsomes at an early stage of drug development

Abstract

1. We established a mechanism-based inhibition cocktail-substrate assay system using human liver microsomes and drug–probe substrates that enabled simultaneous estimation of the inactivation of main cytochrome P450 (CYP) enzymes, CYP2C9, CYP2D6, and CYP3A, in drug metabolism.

2. The inactivation kinetic parameters of typical mechanism-based inhibitors, tienilic acid, paroxetine, and erythromycin, for each enzyme in the cocktail-substrate assay were almost in agreement with the values obtained in the single-substrate assay.

3. Using this system, we confirmed that multiple CYP inactivation caused by mechanism-based inhibitors such as isoniazid and amiodarone could be detected simultaneously.

4. Mechanism-based inhibition potency can be estimated by the determination of the observed inactivation rate constants (k_obs) at a single concentration of test compounds because the k_obs of eleven CYP3A inactivators at 10 μM in the assay system nearly corresponded to k_inact/K_I values, an indicator of a compound’s propensity to alter the activity of a CYP in vivo (R² = 0.97).

5. Therefore, this cocktail-substrate assay is considered to be a powerful tool for evaluating mechanism-based inhibition at an early stage of drug development.

Keywords::

• Cytochrome P450
• mechanism-based inhibition
• cocktail substrate
• human liver microsomes
• drug-drug interaction

Acknowledgements

The authors thank Hiroshi Ito for his technical assistance.

Declaration of interest: The authors report no conflicts of interest.