Journal: Drug Metabolism and Disposition

Article Title: Identification and Characterization of CINPA1 Metabolites Facilitates Structure-Activity Studies of the Constitutive Androstane Receptor

doi: 10.1124/dmd.116.071993

Figure Lengend Snippet: Metabolite formation in HLMs in the presence of specific P450 enzyme inhibitors. Specific inhibitors of various P450 isoforms (or DMSO control) were preincubated at the indicated concentrations with HLMs (0.5 mg/ml) and an NADPH regenerating system at 37°C for 15 minutes. (A and B) 5 µM CINPA1 (A) or Met1 (B) was added to the reaction mixtures, which were then incubated for an additional 30 minutes at 37°C. The reactions were terminated with acetonitrile and analyzed by LC/MS. The enzyme activity was calculated by measuring the amount of metabolite (Met1 or Met2) formed. (C) An esterase inhibitor, BNPP (250 µM), or DMSO (control) was preincubated with HLMs and an NADPH regenerating system at 37°C for 15 minutes as described earlier. Met1 (5 µM) was added to this mixture as the substrate, and the reaction was terminated after an additional 30 minutes of incubation at 37°C. The percent enzyme activity was calculated by normalizing the amount of metabolite formed in the absence of the inhibitor (DMSO) to 100%. Each bar represents the average of triplicate reaction wells ± S.D. One-way analysis of variance was used to compare the percent enzyme activity in the presence of each inhibitor to DMSO control. *P < 0.05. Unmarked bars indicate no significant change observed. The inhibitors used were ketoconazole (KTZ; CYP3A4), quinidine (Quin; CYP2D6), ticlopidine (Ticlo; CYP2C19), sulfaphenazole (SFZ; CYP2C9), quercetin (Querc; CYP2C8), thioTEPA (TT; CYP2B6), PCPA (CYP1A2), and BNPP (esterase). ns, not significant.

Article Snippet: Substrates, metabolite standards, internal standards, inhibitors, and other materials were obtained from the following sources: all anhydrous solvents, dimethylsulfoxide (DMSO), ketoconazole, N , N ′, N ′′-triethylenethiophosphoramide (thioTEPA), ticlopidine, quinidine, efavirenz, tolbutamide, phenacetin, bis( p -nitrophenyl) phosphate (BNPP), and acetaminophen (Sigma-Aldrich, St. Louis, MO); tranylcypromine (PCPA), quercetin, dextromethorphan, 1′-hydroxymidazolam, dextrorphan, hydroxytolbutamide, and (±)4-hydroxymephenytoin (Cayman Chemical, Ann Arbor, MI); sulfaphenazole, midazolam, and the NADPH regenerating system (Corning, Tewksbury, MA); S -mephenytoin (Santa Cruz Biotechnology, Dallas, TX); and 8-hydroxyefavirenz (Toronto Research Chemicals, Toronto, Canada).

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: Drug Metabolism and Disposition

Article Title: Identification and Characterization of CINPA1 Metabolites Facilitates Structure-Activity Studies of the Constitutive Androstane Receptor

doi: 10.1124/dmd.116.071993

Figure Lengend Snippet: Metabolite formation in HLMs in the presence of specific P450 enzyme inhibitors. Specific inhibitors of various P450 isoforms (or DMSO control) were preincubated at the indicated concentrations with HLMs (0.5 mg/ml) and an NADPH regenerating system at 37°C for 15 minutes. (A and B) 5 µM CINPA1 (A) or Met1 (B) was added to the reaction mixtures, which were then incubated for an additional 30 minutes at 37°C. The reactions were terminated with acetonitrile and analyzed by LC/MS. The enzyme activity was calculated by measuring the amount of metabolite (Met1 or Met2) formed. (C) An esterase inhibitor, BNPP (250 µM), or DMSO (control) was preincubated with HLMs and an NADPH regenerating system at 37°C for 15 minutes as described earlier. Met1 (5 µM) was added to this mixture as the substrate, and the reaction was terminated after an additional 30 minutes of incubation at 37°C. The percent enzyme activity was calculated by normalizing the amount of metabolite formed in the absence of the inhibitor (DMSO) to 100%. Each bar represents the average of triplicate reaction wells ± S.D. One-way analysis of variance was used to compare the percent enzyme activity in the presence of each inhibitor to DMSO control. *P < 0.05. Unmarked bars indicate no significant change observed. The inhibitors used were ketoconazole (KTZ; CYP3A4), quinidine (Quin; CYP2D6), ticlopidine (Ticlo; CYP2C19), sulfaphenazole (SFZ; CYP2C9), quercetin (Querc; CYP2C8), thioTEPA (TT; CYP2B6), PCPA (CYP1A2), and BNPP (esterase). ns, not significant.

Article Snippet: Substrates, metabolite standards, internal standards, inhibitors, and other materials were obtained from the following sources: all anhydrous solvents, dimethylsulfoxide (DMSO), ketoconazole, N , N ′, N ′′-triethylenethiophosphoramide (thioTEPA), ticlopidine, quinidine, efavirenz, tolbutamide, phenacetin, bis( p -nitrophenyl) phosphate (BNPP), and acetaminophen (Sigma-Aldrich, St. Louis, MO); tranylcypromine (PCPA), quercetin, dextromethorphan, 1′-hydroxymidazolam, dextrorphan, hydroxytolbutamide, and (±)4-hydroxymephenytoin (Cayman Chemical, Ann Arbor, MI); sulfaphenazole, midazolam, and the NADPH regenerating system (Corning, Tewksbury, MA); S -mephenytoin (Santa Cruz Biotechnology, Dallas, TX); and 8-hydroxyefavirenz (Toronto Research Chemicals, Toronto, Canada).

Techniques: Control, Incubation, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Metabolism and Bioactivation of Corynoline With Characterization of the Glutathione/Cysteine Conjugate and Evaluation of Its Hepatotoxicity in Mice

doi: 10.3389/fphar.2018.01264

Figure Lengend Snippet: Inhibitors and their final concentration for CYP450s enzyme identification.

Article Snippet: Sulfaphenazole (CYP2C9 inhibitor) was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany).

Techniques: Concentration Assay