Substrate / inhibitor influence on CYP2C8 with implications for related isoforms
Compounds were hydroxylated in incubations containing CYP1A2, CYP2B6, or CYP2C19 SupersomesTM, with a large turnover number observed for CYP2B6-dependent flavonone metabolism (>20% loss of parent). These analogues did not appear to be within a critical distance of the haem moiety in CYP2C8 or CYP3A4 substrate-binding sites as evidenced by lack of metabolism. The paclitaxel precursor, baccatin III was also demonstrated not to be a CYP2C8 substrate, with levels of a potentially 6-hydroxylated metabolite being undetectable by LC-MS/MS analyses. Therefore, a C13 paclitaxel side chain is essential for CYP2C8-dependent 6-hydrodylation. Apigenin was shown to inhibit paclitaxel 6-hydroxylase activity competitively in SupersomesTM with a Ki value of 0.8μM determined. Critical hydrophobic CYP2C8 residues have been proposed that form hydrophobic contacts with rosiglitazone, namely IIE113, Ser114, Phe 205, Ala294, Val363 and Val474. Assuming that only a limited number of residues project into the substrate-binding site, it is likely that apigenin and paclitaxel bind mutually exclusively with one or more of these residues within this vicinity. A Km/Ki ration of 2.5 indicates a preference of active site for inhibitor. Apigenin also inhibited CYP2C8-dependent benzo(a)pyrene 3-hydroxylation in SupersomesTM and CYP3A4-dependent felodipine dehydrogenation, CYP2C9-dependent tolbutamide hydroxylation and CYP2C19-dependent omeprazole 5-hydroxylation in HLMs or SupersomesTM. Non-competitive modes of apigenin inhibition were established for CYP2C9 and CYP2C19 in SupersomesTM, with Ki values of 5 and 7μM, respectively. Liganding of the hydroxyl group at position 7 with the haem iron may account for non-competitive interaction. Binding spectra data are required to confirm this. Substitution of 4’ methoxyl group (acacetin) instead of hydroxyl group in this position (apigenin) removed CYP2C9 inhibition potential, while CYP2C8 and CYP2C19 inhibition potential were maintained. This implies unique CYP2C9 residues. This is supported by CYP2C9 being reported to display substrate selectivity within CYP2C for flavon-3-ol compounds. Activation of CYP3A4-dependent felodipine dehydrogenation was observed by non-hydroxylated flavonoids in HLMs and SupersomesTM. Preliminary evidence suggests that CYP2C8 may also contain an effector site, with 10 μM acacetin stimulating CYP2C8-dependent benzo(a)pyrene 3-hydroxylation to 188% of control activity. Lipophilic and aromatic CYP2C8 interactions are essential for substrate/inhibitor interactions. This is supported by physicochemical data, kinetic evaluation of CYP2C8-dependent activity and flavonoid inhibition data. Predominance of aromatic groups in these high affinity substrates and inhibitors reveal that CYP2C8 hydrophobic interactions are important.