The present results warrant a further evaluation of BBRCdrug interaction in clinical situations and a determination of inter-individual variability in CYP2D6 inhibition by BBR, based on genetic polymorphism of CYP2D6. 5. as in the case of BBR, which suggests that methylenedioxybenzene moiety of BBR may play a critical role in the quasi-irreversible inhibition. Moreover, the metabolic clearance of nebivolol (-blocker; CYP2D6 substrate) was reduced in the presence of BBR. The present results warrant further evaluation of BBRCdrug interactions in clinical situations. (see Supplementary Materials). Pooled HLM and recombinant human CYP2D6 (rhCYP2D6; product number: 456217) were obtained from Corning Gentest (Corning, NY, USA) Alpha-Naphthoflavone and Alpha-Naphthoflavone stored at ?80 C before use. All the other chemicals and reagents used were of analytical grade. 2.2. Direct CYP Inhibition Assay Using Pooled HLM The CYP inhibition assay was conducted as described previously [29,30]. In brief, BBR or its analogue (THB, TRB, BRB, TFD, DMB, or JTZ) at various concentrations (up to 100 M) was pre-incubated with pooled HLM (0.2 mg/mL) and two different CYP isoform-selective substrate cocktail sets (set A: 50 M of phenacetin, 5 M of coumarin, 0.2 M of amodiaquine, 100 M of values at various inhibitor concentrations Equation (1) : for 20 min at 4 Alpha-Naphthoflavone C, and the supernatants were analyzed by the LC-MS/MS system described in Section 2.2. The sample injection volume was 5 L, and the separation was performed on an XTerra?MS C18 column (2.1 150 mm, 5 m; Waters, Milford, MA, USA). The mobile phases consisted of DDW containing 0.1% (for 20 min at 4 C. The supernatants were analyzed using the same LC-MS/MS method described in Section 2.2, except that a Shimadzu 20AD-XR HPLC system (Shimadzu, Kyoto, Japan) was employed for the chromatographic separation of analytes. The mass transitions of nebivolol and CBZ were 406 to 151 and 237 to 194, KRT20 respectively. 2.8. Data Analysis In vitro metabolic stability parameters, including half-life (represents the first-order degradation rate constant. The values were Alpha-Naphthoflavone estimated by fitting a one-phase exponential decay model to the parent drug remaining (%) vs. incubation time plots using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). The was determined using the in vitro half-life approach. The was normalized by the concentration of microsomal protein used (mg/L), as displayed in the following Equation (3): of BBR was calculated to be 7.82 L/min/mg protein, suggesting that BBR is moderately stable in HLM. The formation of BBR metabolites was monitored over the 120 min of incubation with HLM (Table 1), in which the identification and quantification of metabolites were performed simultaneously using authentic standards, including BRB, TFD, DMB, and JTZ. As the incubation time elapsed, the concentrations of TFD, DMB, and M1 (an unknown metabolite observed at 310) in the reaction mixture increased gradually. BRB exhibited a slight increase in its concentration, whereas JTZ showed no difference. M1 was further characterized using an accurate-MS system (Supplementary Materials, Table S2). The proposed chemical formula of M1 is C18H16NO4+ with a mass error of 1 1.9 ppm, and the fragment ions of M1 were observed at 295.0824 and 267.0890. Notably, M1 was also generated from the incubation of TFD or DMB with HLM in the presence of NADPH, but not in the absence of NADPH (Supplementary Materials, Table S3), suggesting that M1 could be produced from BBR via TFD or DMB. The product ions of M1 were observed at 295 and 267 (Supplementary Materials, Table S2), showing a difference of 12 mass units compared to the product ions of TFD (307 and 279) and a difference of 14 mass units compared to the product ions of DMB (309). These results suggest that M1 could be demethylene-TFD. Table 1 Elimination of BBR and formation of its metabolites in pooled HLM. = 310)and using the relationship between and inhibitor concentrations (Figure 4). The estimated values of and of BBR against CYP2D6 were 0.025 min?1, 4.29 M and 5.83 mL/min/mol, respectively. Open in a separate window Figure 4 Time-dependent inhibition of CYP2D6 by BBR. (A) BBR was incubated for 0, 1, 5, 10, 15, or 30 min with NADPH in pooled HLM. CYP2D6 activity is expressed as the log percentage of control group containing.