Zhong-Hua Chen1,2, Su-Xing Zhang3, Na Long3, Li-Shan Lin3, Tao Chen3, Fei-Peng Zhang3, Xue-Qin Lv3, Pei-Zhen Ye3, Ning Li4, Ke-Zhi Zhang3. 1. Department of Pharmacology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China. 2. Key Laboratory of Drug Targeting, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China; 3. CarysBio Holdings Limited, Chancheng, Foshan 528000, China. 4. School of Biological Science and Technology, University of Ji-nan, Ji-nan 250022, China.
Abstract
AIM: 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. CONCLUSION: 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.
AIM: 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. CONCLUSION: 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: Scott W Grimm; Heidi J Einolf; Steven D Hall; Kan He; Heng-Keang Lim; Kah-Hiing John Ling; Chuang Lu; Amin A Nomeir; Eleanore Seibert; Konstantine W Skordos; George R Tonn; Robert Van Horn; Regina W Wang; Y Nancy Wong; Tian J Yang; R Scott Obach Journal: Drug Metab Dispos Date: 2009-04-09 Impact factor: 3.922
Authors: J Andrew Williams; Ruth Hyland; Barry C Jones; Dennis A Smith; Susan Hurst; Theunis C Goosen; Vincent Peterkin; Jeffrey R Koup; Simon E Ball Journal: Drug Metab Dispos Date: 2004-08-10 Impact factor: 3.922