proton pump inhibitor When six chemical inhibitors were incu
When six chemical inhibitors were incubated with TRB or TRC and human liver microsomal preparation, HLM2, the results showed that ketoconazole (a CYP3A4 inhibitor) was very effective in inhibiting 4β-C hydroxylation of TRB and TR, whereas sulfaphenazole (a CYP2C9 inhibitor) and quinidine (a CYP2D6 inhibitor) caused less inhibition (Fig. 1). Concentration–response curves for the effect of ketoconazole, sulfaphenazole, or quinidine on metabolism by preparation HLM2 suggested that CYP2D6 and CYP3A4 were involved in MB2, MB4, and MC formation from TRB and TRC (Fig. 2, Fig. 3, Fig. 4). However, studies with HLM2 and HLM3 and the goat anti-CYP3A4 antibody, which also blocks CYP3A5 and CYP3A7, showed that the antibody could completely block the formation of MB2, MB4, and MC (Fig. 5). The study on supersomes from baculovirus-transformed insect proton pump inhibitor expressing different human CYP450 isoforms demonstrated that CYP3A4 and CYP3A5 had TRB and TRC 4β-C hydroxylation activity and TRB O-demethylation activity, while CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 did not (Table 1). Furthermore, the experiment with CYP3A4-expressing V79MZh3A4 cells confirmed that CYP3A4 was able to form MB2 and MB4 from TRB and MC from TRC (Table 1). Further experiments (Table 5) confirmed the correlation between the formation of TRB or TRC metabolites and 6β-testosterone hydroxylase activity (r2=0.99, P<0.0001 for MB2, r2=0.95, P<0.0001 for MB4, and r2=0.97, P<0.0001 for MC). Together, the above results suggest that MB2, MB4, and MC formation is mediated by CYP3A4 and CYP3A5. In rats, gender-specific CYP3A isoforms (Kobliakov et al., 1991) and gender differences in TRA metabolism (Peng and Lin Wu, 2002, Lin Wu et al., 2003) have been reported. In the present study on humans, microsomal preparations from males (HLM1, HLM4, HH40, HG64, and HG103) and females (HLM2, HLM3, HH18, HH31, HG3, and HG88) of different ages showed the same profile of TRB and TRC metabolites (Table 1). Gender-specific CYP3A enzymes have not been described in humans, and Hunt et al. (1992) reported that CYP3A4 does not exhibit marked gender differences in expression. A 10–100-fold difference between individuals in CYP3A4 activity in humans has been reported (Shimada et al., 1994). Our human liver microsome preparations showed differences in TRB 4β-C hydroxylation activity (0.40–26.05nmolmin−1mg−1protein), TRB O-demethylation (0.40–26.05nmolmin−1mg−1protein), TRB 4β-C hydroxylation activity (0.26–1.31nmolmin−1mg−1protein), and 6β-testosterone hydroxylase activity (0.36–8.69pmolhydroxytestosteronemin−1mg−1protein). The amount of MB2 and MB4 and MC formed from TRB and TRC by these HLM preparations was related to the 6β-testosterone hydroxylase activity (Table 1, Table 6). CYP3A4 and CYP3A5 protein and mRNA were expressed in all four human liver microsome preparations tested (HLM1–HLM4) (Fig. 6). In general, for most CYP3A substrates for hydroxylation, the in vitro intrinsic clearance for CYP3A4 is greater than that for CYP3A5 (Williams et al., 2002, Patki et al., 2003, Cook et al., 2002, Kalgutkar et al., 2003, Shen et al., 2004). When 4β-C hydroxylation and O-demethylation of TRB was performed using supersomes expressing CYP3A4 or CYP3A5, the two isoforms showed comparable activity (Table 1), but the intrinsic clearance of 4β-C hydroxylation of TRB was higher for CYP3A4 than for CYP3A5, while that for the O-demethylation of TRB was higher for CYP3A5 (Fig. 7). This suggests that 4β-C hydroxylation of TRB is catalyzed mainly by CYP3A4 and that O-demethylation of TRB is catalyzed mainly by CYP3A5. CYP3A5 shares considerable sequence homology with, and has qualitatively similar substrate selectivity to CYP3A4. However, the relative metabolic activities of CYP3A4 and CYP3A5 are substrate-dependent and regioselective (Person et al., 1997, Xie et al., 2004). Although the amino acid sequences of CYP3A4 and CYP3A5 are 84% identical, their functional differences indicate that key differences may exist in their active sites. Key differences can be seen in amino acid residues in substrate recognition sites (SRS) regions in CYP3A4 and CYP3A5 that confer functional competence and/or divergence, novel catalytic specificities and activities, and specific regio- and stereo-selective targeting of a given substrate (Wang et al., 1998). Thus, the SRS differences between CYP3A4 and CYP3A5 appear to critically influence the metabolism of TRB. Recently, several prediction methods, including relative activity factors (RAFs), for assessing the contribution of multiple CYPs to certain metabolic reactions in human liver microsomes have been reported (Crespi, 1995, Iwatsubo et al., 1997, Becquemont et al., 1998). In a future study based on RAFs, we will estimate the contributions of CYP3A4 and CYP3A5 to the hydroxylation and O-demethylation of TRB in human liver microsomes using recombinant CYP3A4 and CYP3A5 expressed in baculovirus-transformed insect cells.