title: An improved substrate cocktail for assessing direct inhibition and time-dependent inhibition of multiple cytochrome P450s.
abstract: OBJECTIVE
  The substrate cocktail is frequently used to evaluate cytochrome P450 (CYP) enzyme-mediated drug interactions and potential interactions among the probe substrates. Here, we re-optimized the substrate cocktail method to increase the reliability and accuracy of screening for candidate compounds and expanded the method from a direct CYP inhibition assay to a time-dependent inhibition (TDI) assay.
  
  
  METHODS
  In the reaction mixtures containing human liver microsome (0.1 mg/mL), both the concentrations of a substrate cocktail (phenacetin for 1A2, coumarin for 2A6, bupropion for 2B6, diclofenac for 2C9, dextromethorphan for 2D6, and testosterone for 3A4) and the incubation time were optimized. Metabolites of the substrate probes were simultaneously analyzed by multiple-reaction monitoring (MRM) using a routine LC/MS/MS. Direct CYP inhibition was validated using 7 inhibitors (α-naphthoflavone, tranylcypromine, ticlopidine, fluconazole, quinidine, ketoconazole and 1-ABT). The time-dependent inhibition was partially validated with 5 inhibitors (ketoconazole, verapamil, quinidine, paroxetine and 1-ABT).
  
  
  RESULTS
  The inhibition curve profiles and IC50 values of 7 CYP inhibitors were approximate when a single substrate and the substrate cocktail were tested, and were consistent with the previously reported values. Similar results were obtained in the IC50 shifts of 5 inhibitors when a single substrate and the substrate cocktail were tested in the TDI assay.
  
  
  CONCLUSIONS
  The 6-in-1 substrate cocktail (for 1A2, 2A6, 2B6, 2C9, 2D6 and 3A) is reliable for assessing CYP inhibition and time-dependent inhibition of drug candidates.
authors.affiliation: Department of Pharmacology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China.|Key Laboratory of Drug Targeting, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China;
authors.forename: Zhong-Hua
authors.initials: ZH
authors.lastname: Chen
authors.affiliation: CarysBio Holdings Limited, Chancheng, Foshan 528000, China.
authors.forename: Su-Xing
authors.initials: SX
authors.lastname: Zhang
authors.affiliation: CarysBio Holdings Limited, Chancheng, Foshan 528000, China.
authors.forename: Na
authors.initials: N
authors.lastname: Long
authors.affiliation: CarysBio Holdings Limited, Chancheng, Foshan 528000, China.
authors.forename: Li-Shan
authors.initials: LS
authors.lastname: Lin
authors.affiliation: CarysBio Holdings Limited, Chancheng, Foshan 528000, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
authors.affiliation: CarysBio Holdings Limited, Chancheng, Foshan 528000, China.
authors.forename: Fei-Peng
authors.initials: FP
authors.lastname: Zhang
authors.affiliation: CarysBio Holdings Limited, Chancheng, Foshan 528000, China.
authors.forename: Xue-Qin
authors.initials: XQ
authors.lastname: Lv
authors.affiliation: CarysBio Holdings Limited, Chancheng, Foshan 528000, China.
authors.forename: Pei-Zhen
authors.initials: PZ
authors.lastname: Ye
authors.affiliation: School of Biological Science and Technology, University of Ji-nan, Ji-nan 250022, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Li
authors.affiliation: CarysBio Holdings Limited, Chancheng, Foshan 528000, China.
authors.forename: Ke-Zhi
authors.initials: KZ
authors.lastname: Zhang
mesh_terms: D065607:Cytochrome P-450 Enzyme Inhibitors / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004347:Drug Interactions; D006801:Humans; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System