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.

Activity and mRNA expression levels of selected cytochromes P450 in various sections of the human small intestine

“…Two chromatographic analytical methods were used for the metabolites of: (i) bupropion, ebastine and midazolam; and (ii) repaglinide, tolbutamide, S‐mephenytoin, bufuralol, chlorzoxazone and lauric acid as we described previously . Chromatographic conditions are presented in Supplemental Methods (Appendix 1) and Table S2 (Appendix 2).…”

A Pilot Study towards the Impact of Type 2 Diabetes on the Expression and Activities of Drug Metabolizing Enzymes and Transporters in Human Duodenum

“…Specificity of the probe substrates in the intestinal biopsies was evaluated using selective inhibitors: 10 µM ticlopidine (CYP2B6), 10 µM sulfaphenazole (CYP2C9), 150 µM astemizole (CYP2J2), and 5 µM ketoconazole (CYP3A4/5). Following incubation, the formation rate of metabolites was determined by measuring their concentrations using a sensitive and precise LC-MSMS detection method [12,88]. Activity was normalized for total protein content measured using the Pierce ® BCA Protein Assay Kit (ThermoFisher Scientific Inc., USA).…”

Use of 4β‐Hydroxycholesterol Plasma Concentrations as an Endogenous Biomarker of CYP3A Activity: Clinical Validation in Individuals With Type 2 Diabetes

“…Its selectivity allows for its use alone as well as in a combination with other probe drugs to determine simultaneously several CYP450 phenotypes. [13][14][15][16][17][18] In addition to their role in the metabolism of drugs, CYP450s are also involved in redox reactions of endogenous substances, as they participate in 94% of important homeostatic processes. 19 As for drug metabolism, CYP3As could be involved in 18% of those reactions.…”

“…The probe substrate midazolam has been widely used and validated as the gold standard for phenotyping CYP3A activity in vivo . Its selectivity allows for its use alone as well as in a combination with other probe drugs to determine simultaneously several CYP450 phenotypes …”