Development and validation of probe drug cocktails for the characterization of CYP450-mediated metabolism by human heart microsomes

Abstract

1. The objective of our study was to develop and validate a cocktail approach to allow the simultaneous characterization of various CYP450-mediated oxidations by human heart microsomes for nine probe drug substrates, namely, 7-ethoxyresorufin, bupropion, repaglinide, tolbutamide, bufuralol, chlorzoxazone, ebastine, midazolam and dodecanoic acid.

2. The first validation step was conducted using recombinant human CYP450 isoenzymes by comparing activity measured for each probe drug as a function of (1) buffer used, (2) selectivity towards specific isoenzymes and (3) drug interactions between probes. Activity was all measured by validated LC-MSMS methods.

3. Two cocktails were then constituted with seven of the nine drugs and subjected to kinetic validation. Finally, all probe drugs were incubated with human heart microsomes prepared from ventricular tissues obtained from 12 patients undergoing cardiac transplantation.

4. Validated cocktail #1 including bupropion, chlorzoxazone, ebastine and midazolam was used to characterize CYP2B6-, 2E1-, 2J2- and 3A5-mediated metabolism in human hearts.

5. Cocktail #2 which includes bufuralol, 7-ethoxyresorufin and repaglinide failed the validation step. Substrates in cocktail #2 as well as tolbutamide and dodecanoic acid had to be incubated separately because of their physico-chemical characteristics (solubility and ionization) or drug interactions.

6. Activity in HHM was the highest towards ebastine, chlorzoxazone and tolbutamide.

Keywords:

• CYP450
• cocktail
• probe
• metabolism
• recombinant
• microsome
• LC-MSMS

Disclosure statement

J. Huguet, F. Gaudette, V. Michaud and J. Turgeon report no conflicts of interest.

Additional information

Funding

This work was supported by the Canadian Institutes of Health Research (CIHR) (268829) and internal funding obtained from the Fondation du CHUM. Jade Huguet was subsequently the recipient of a studentship from the CIHR (203132) and the Fonds de Recherche Santé Québec (FRSQ) (25436). Veronique Michaud is the recipient of a research scholarship from FRQS in partnership with the Institut national d’excellence en santé et en services sociaux (INESSS) (28970).