To further confirm the roles of the
To further confirm the roles of the CYP450 enzymes involved in the metabolism of Dip in rat liver microsomes, we utilized not only chemical inhibitors but also a correlation analysis and a panel of recombinant rat CYP450 enzymes to evaluate the contribution of CYP450 enzymes. The results of the correlation analysis were consistent with the conclusions from the chemical inhibition experiments (Table 2). Incubation of Dip with nine recombinant rat CYP450 enzymes also showed that CYP2A1 and CYP3A were the major enzymes responsible for the generation of four metabolites, and of the two CYP3A isoenzymes, CYP3A2 exhibited a greater contribution than CYP3A1 (Fig. 6). The results were also similar to those from the chemical inhibition experiments and the correlation analysis.
The high agreement of results from the chemical inhibition study, the correlation analysis and the recombinant rat CYP450 enzymes further confirmed the reliability of our conclusion in the present study. However, in Brefeldin A to the chemical inhibition experiments and correlation analysis, the study involving recombinant rat CYP450 enzymes found that trace amounts of the four metabolites were generated by CYP2B1 and CYP2D1. This phenomenon can be explained by the tendency of a recombinant CYP450 enzyme to demonstrate metabolism activity in the absence of other CYP450 enzymes, and it may play little or no role in the microsomal metabolism in the presence of other CYP450 isoforms due to the competitive nature of the CYP450 enzymes (Tucker et al., 2001).
If multiple CYP450 enzymes are involved in a drug's metabolism, their kinetics must be studied to assess their contribution to the hepatic clearance of the drugs. Therefore, we examined the enzyme kinetic parameters of the formation of four Dip metabolites by rat liver microsomes and recombinant rat CYP3A1 and CYP3A2. As shown in Table 1, Table 3, the Km for the formation of metabolites by rat liver microsomes is similar to those by recombinant rat CYP3A1 and CYP3A2. The comparable Km values strongly suggest that CYP3A is a major contributor to the generation of the metabolite. The contribution of CYP2A1 is likely less than that of CYP3A.
The contribution of CYP450 enzymes to the metabolism of other diphenylpiperazine calcium blockers, such as CZ and FZ, had been previously reported in rats, dogs and humans (Meuldermans et al., 1983, Kariya et al., 1992, Kariya et al., 1996, Narimatsu et al., 1993, Lavrijsen et al., 1992). The hydroxylation of the aromatic ring and N-dealkylation constituted the primary metabolic pathways for the microsomal metabolism of CZ and FZ (Narimatsu et al., 1993). Among the four examined metabolites of Dip, M1 and M5 are the products of N-dealkylation, and M2 is a product of aromatic ring hydroxylation. However, the CYP450 enzymes involved in the processing of Dip do not exactly match those described in the literature above. For instance, CYP2B6 was involved in the p-hydroxylation of CZ when CZ and FZ were examined in microsomes from lymphoblastoid cells that expressed human CYP450 enzymes; however, in our present study, CYP2B1 did not exhibit p-hydroxylation of the diphenylmethyl group of Dip (M2 formation). At the same time, the CYP450 enzymes that mediated the 1-N-dealkylation of the piperazine ring of Dip were different from those involved in the 1-N-dealkylation of CZ and FZ (Kariya et al., 1996). The differences in the metabolic profiles might be due to the species-specific differences because our present study was carried out in rats or/and due to the introduction of the second fluorine atom to the cinnamyl phenyl ring of Dip.
Conflict of interest
Acknowledgements This work was supported by the Natural Science Foundation of Hebei Province [Grant C2006001035] and the Technology Supporting Plan of Hebei Province [Grant 10276434]. We thank the Department of Medicinal Chemistry at Hebei Medical University for providing the reference standard samples of Dip and its metabolites.