Development and validation of probe drug cocktails for the characterization of CYP450-mediated metabolism by human heart microsomes.
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.
['Huguet|Jade|J|', 'Gaudette|Fleur|F|', 'Michaud|Veronique|V|', 'Turgeon|Jacques|J|']
D016642:Bupropion D002090:Butyrophenones D002219:Carbamates D002753:Chlorzoxazone D003577:Cytochrome P-450 Enzyme System D004353:Drug Evaluation, Preclinical D004983:Ethanolamines D006801:Humans D007850:Lauric Acids D008861:Microsomes D008874:Midazolam D009206:Myocardium D010078:Oxazines D010880:Piperidines D014044:Tolbutamide
probe cocktail metabolism microsome recombinant CYP450 LC-MSMS