Boehringer Ingelheim Pharmaceuticals Inc., Drug Metabolism and Pharmacokinetics, Ridgefield, Connecticut
Boehringer Ingelheim Pharmaceuticals Inc., Drug Metabolism and Pharmacokinetics, Ridgefield, Connecticut.
Drug Metab Dispos. 2020 Aug;48(8):690-697. doi: 10.1124/dmd.120.090951. Epub 2020 Jun 5.
Long-term hepatocyte culture systems such as HepatoPac are well suited to evaluate the metabolic turnover of low clearance (CL) drugs because of their sustained metabolic capacity and longer-term viability. Erythromycin (ERY), a moderate, mechanism-based inhibitor of CYP3A, was evaluated as a tool in the HepatoPac model to assess contribution of CYP3A to the clearance of drug candidates. ERY inhibited CYP3A activity by 58% and 80% at 3 and 10 μM, respectively, for up to 72 hours. At 30 µM, ERY inhibited midazolam hydroxylation by >85% for the entire 144-hour duration of the incubation. Alprazolam CL was inhibited 58% by 3 μM of ERY, 75% by 15 μM of ERY, 89% by 30 μM of ERY, and 94% by 60 μM of ERY. ERY (30 μM) did not markedly affect CL of substrates for several other major cytochrome P450 isoforms evaluated and did not markedly inhibit uridine diphosphoglucuronosyl transferase (UGT) isoforms 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, or 2B15 as assessed using recombinant UGTs. ERY only mildly increased CYP3A4 gene expression by 2.1-fold (14% of rifampicin induction) at 120 µM, indicating that at effective concentrations for inhibition of CYP3A activity (30-60 µM), arylhydrocarbon receptor, constitutive androstane receptor, and pregnane-X-receptor activation are not likely to markedly increase levels of other drug-metabolizing enzymes or transporters. ERY at concentrations up to 60 µM was not toxic for up to 6 days of incubation. Use of ERY to selectively inhibit CYP3A in high-functioning, long-term hepatocyte models such as HepatoPac can be a valuable strategy to evaluate the contribution of CYP3A metabolism to the overall clearance of slowly metabolized drug candidates. SIGNIFICANCE STATEMENT: This work describes the use of erythromycin as a selective inhibitor of CYP3A to assess the contribution of CYP3A in the metabolism of compounds using long-term hepatocyte cultures.
长期肝细胞培养系统，如 HepatoPac，非常适合评估低清除率（CL）药物的代谢转化，因为它们具有持续的代谢能力和更长的存活时间。红霉素（ERY）是一种中等强度的、基于机制的 CYP3A 抑制剂，被评估为 HepatoPac 模型中的一种工具，以评估 CYP3A 对候选药物清除率的贡献。ERY 在 3 和 10 μM 时分别抑制 CYP3A 活性 58%和 80%，持续 72 小时。在 30 µM 时，ERY 抑制咪达唑仑羟化作用>85%，持续 144 小时孵育。ERY 以 3 μM 抑制 alprazolam 的 CL 为 58%，以 15 μM 抑制 75%，以 30 μM 抑制 89%，以 60 μM 抑制 94%。ERY（30 μM）对评估的几种其他主要细胞色素 P450 同工酶的底物的 CL 没有明显影响，也没有明显抑制尿苷二磷酸葡萄糖醛酸转移酶（UGT）同工酶 1A1、1A3、1A4、1A6、1A9、2B7 或 2B15，如使用重组 UGTs 评估的那样。ERY 仅以 2.1 倍（利福平诱导的 14%）轻度增加 CYP3A4 基因表达，表明在有效抑制 CYP3A 活性的浓度（30-60 µM）下，芳香烃受体、组成型雄激素受体和孕烷 X 受体的激活不太可能显著增加其他药物代谢酶或转运体的水平。ERY 在高达 60 µM 的浓度下在孵育长达 6 天的时间内没有毒性。在高功能、长期肝细胞模型（如 HepatoPac）中使用 ERY 选择性抑制 CYP3A 可以成为一种有价值的策略，用于评估 CYP3A 代谢对缓慢代谢候选药物总体清除率的贡献。意义声明：这项工作描述了使用红霉素作为 CYP3A 的选择性抑制剂，用于评估长期肝细胞培养物中化合物的 CYP3A 代谢对其代谢的贡献。
