In some cases more than

In some cases, more than one CYP450 enzyme may be involved in the metabolism of a drug, and an Eadie–Hofstee plot is usually more reliable for assessing the involvement of multiple bepridil (Bu, 2006). Therefore, we first plotted the Eadie–Hofstee curves and observed their shape to determine the kinetic model of Dip metabolism. The results from the Eadie–Hofstee plots and the chemical inhibition experiments indicated that more than one CYP450 enzyme was responsible for the metabolism of Dip. Interestingly, CYP2A1, CYP2C11 and CYP3A were involved in the formation of all of the metabolites. In these processes, Dip was mainly metabolized by CYP2A1 and CYP3A and CYP2C11 played only a minor role. CYP2B1, CYP2C6 and CYP2D1 were not involved in the metabolism of Dip. Our recent rat liver microsome research demonstrated that Dip undergoes 1-N-dealkylation that leads to the formation of M1 and M5 and then M5 is oxidized or reduced to M2 and M4, respectively, while M1 is converted to M3 via 4-N-dealkylation and γ-hydroxylation (Guo et al., 2012). Because M1 and M5 were simultaneously produced as a result of 1-N-dealkylation of the diphenylpiperazine portion of the molecule, the CYP450 enzymes that metabolize Dip to M1 and M5 were thought to be identical. Indeed, the results of the present study are consistent with our presumption that the CYP450 enzymes involved in the formation of M1 and M5 are identical (CYP2A1, CYP3A and CYP2C11). Moreover, the chemical inhibition study also showed that M2 formation could be inhibited by diethyldithiocarbamate and α-naphthoflavone and the formation of M4 could be inhibited by diethyldithiocarbamate. This suggested that, in addition to CYP2A1, CYP3A and CYP2C11, CYP1A2 and CYP2E1 were also involved in the formation of M2, and CYP2E1 was also responsible for M4 formation. However, because M2 and M4 are generated from M5 and not directly from Dip, it is necessary to further study whether CYP2A1, CYP3A and CYP2C11 are directly involved in the formation of M2 and M4 from M5 or indirectly affect their formation by metabolizing Dip to M5. The chemical inhibition experiments showed that three or more CYP450 enzymes were involved in the rat microsomal metabolism of Dip. The reason for this observation may be due to the use of the different and limited numbers of rat liver samples and/or because of the nonspecificity of the inhibitors for individual CYP450 enzyme. However, the effect of the individual differences between or the limited number of the rat liver samples was negligible because pooled rat liver microsomes from 15 rats were used to identify the CYP450 enzymes in the above experiments. An ideal chemical inhibitor should inhibit only a single CYP450 enzyme. However, some chemical inhibitors can inhibit several CYP450 enzymes simultaneously or an inhibitor at very high concentration may inhibit multiple CYP450 enzymes. In the present inhibition experiments, the specific chemical inhibitors for respective CYP isoform were selected and the inhibitors concentrations were in a low range to avoid cross-reactivity (Wang et al., 2008, Liu et al., 2006, Liu et al., 2011, Kimonen et al., 1995, Wu et al., 2003, Kobayashi et al., 2003, Chang et al., 1992, Levine and Bellward, 1995). Based on the results of the chemical inhibitors, the present study found that pilocarpine and ketoconazole showed the most inhibition and cimetidine showed minor inhibition of the formation of all of the metabolites, but orphenadrine, sulfaphenazole and quinidine did not inhibit the generation of any metabolite. Moreover, α-naphthoflavone inhibited M2 formation and diethyldithiocarbamate inhibited the formation of M2 and M4. These results also indicated that Dip was mainly metabolized by CYP2A1 and CYP3A, while CYP2C11 played only a minor role in these processes, whereas CYP2B1, CYP2C6 and CYP2D1 were not involved in the metabolism of Dip. CYP1A2 and CYP2E1 showed minor contributions; the former was only involved in M2 formation, and the latter could contribute to the formation of M2 and M4. These data support the conclusion made from their inhibition profiles. The results indicated that these inhibitors are as selective for the CYP450 enzymes rat liver microsomes as in human liver microsomes.