Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA.
Drug Metab Dispos. 2011 Jun;39(6):1054-7. doi: 10.1124/dmd.111.038208. Epub 2011 Mar 10.
A refined cytochrome P450 (P450) enzyme IC₅₀ shift assay for more accurately screening CYP3A time-dependent inhibitors (TDIs) is presented. In contrast to the regular IC₅₀ shift assay, in which only one pair of P450 inhibition curves is generated, this modified method generates two pairs of inhibition curves; one pair of curves is created from human liver microsomal incubations with the test article in the presence or absence of NADPH (curves 1 and 2) (same as the traditional assay), and the other pair is created from new microsomal incubations with extract (compound/metabolites) of previous incubations (curves 3 and 4). To assess the true CYP3A time-dependent inhibition, we propose a new parameter, the vertical IC₅₀ curve shift (VICS), represented by vertical shift difference between the two sets of curves divided by inhibitor concentration at which maximal vertical shift of curves 1 and 2 is observed. A shift in the curves 1 and 2 could mean a time-dependent inhibition or formation of a more active inhibitory metabolite(s). The new method provides more reliable characterization of the shift as a result of a true TDI- or metabolite-mediated reversible inhibition. Nine known TDI drugs were evaluated using this refined shift assay. The derived VICS values correlated well with the reported k(inact)/K(I) values derived via the conventional dilution assay method. Thus, the refined assay can be used to identify a true TDI and quantitatively assess the inactivation potential of TDIs in a high-throughput fashion. This assay can be invaluable to screen for true P450 TDIs in the early drug discovery.
本文提出了一种改良的细胞色素 P450（CYP）酶 IC₅₀ 偏移测定法，用于更准确地筛选 CYP3A 时间依赖性抑制剂（TDI）。与常规的 IC₅₀ 偏移测定法不同，后者仅生成一对 CYP 抑制曲线，该改良方法生成两对抑制曲线；一对曲线来自于用人肝微粒体孵育，在存在或不存在 NADPH 的情况下测试药物（曲线 1 和 2）（与传统测定法相同），另一对曲线来自于新的微粒体孵育，使用前孵育的提取物（化合物/代谢物）（曲线 3 和 4）。为了评估真正的 CYP3A 时间依赖性抑制，我们提出了一个新的参数，即垂直 IC₅₀ 曲线偏移（VICS），由两对曲线之间的垂直偏移差异表示，除以观察到曲线 1 和 2 的最大垂直偏移的抑制剂浓度。曲线 1 和 2 的偏移可能意味着时间依赖性抑制或形成更活跃的抑制性代谢物。由于真正的 TDI 或代谢物介导的可逆抑制，新方法提供了对偏移的更可靠特征描述。使用该改良的偏移测定法评估了 9 种已知的 TDI 药物。得出的 VICS 值与通过常规稀释测定法得出的报告的 k(inact)/K(I)值很好地相关。因此，该改良的测定法可用于识别真正的 TDI，并以高通量的方式定量评估 TDI 的失活潜力。在早期药物发现中，该测定法对于筛选真正的 P450 TDI 非常有价值。
