Rifampicin is a strong inducer of cytochrome P450 (CYP3A4) and P-glycoprotein (P-gp/ABCB1), leading to profound drug–drug interactions. In contrast, the chemically related rifabutin does not show such pronounced induction properties in vivo. The aim of our study was to conduct a comprehensive analysis of the different induction potentials of rifampicin and rifabutin in primary human hepatocytes and to analyze the mechanism of potential differences. Therefore, we evaluated CYP3A4/ABCB1 mRNA expression (polymerase chain reaction), CYP3A4/P-gp protein expression (immunoaffinity–liquid chromatography–mass spectrometry, IA-LC-MS/MS), CYP3A4 activity (testosterone hydroxylation), and considered intracellular drug uptake after treatment with increasing rifamycin concentrations (0.01–10 µM). Furthermore, rifamycin effects on the protein levels of CYP2C8, CYP2C9, and CYP2C19 were analyzed (IA-LC-MS/MS). Mechanistic analysis included the evaluation of possible suicide CYP3A4 inhibition (IC₅₀ shift assay) and drug impact on translational efficiency (cell-free luminescence assays). Rifabutin accumulated 6- to 15-fold higher in hepatocytes than rifampicin, but induced CYP3A4 mRNA comparably to rifampicin (e. g. rifampicin 61-fold vs. rifabutin 44-fold, 72 h). While rifampicin for example enhanced protein (10 µM: 21-fold) and activity levels considerably (53-fold), rifabutin only slightly increased CYP3A4 protein expression (10 µM: 3.3-fold) or activity (11-fold) compared to rifampicin after 72 h. Both rifamycins similarly influenced expression of other eliminating proteins. A potential CYP3A4 suicide inhibition by a specific rifabutin metabolite or disruption of ribosome function were excluded experimentally. In conclusion, the lack of protein enhancement, could explain rifabutin’s weaker induction-related drug–drug interaction risk in vivo.

利福平是细胞色素 P450 (CYP3A4) 和 P-糖蛋白 (P-gp/ ABCB1 ) 的强诱导剂，可导致深刻的药物间相互作用。相反，化学相关的利福布汀在体内没有表现出如此明显的诱导特性。本研究的目的是全面分析利福平和利福布丁在人原代肝细胞中的不同诱导电位，并分析电位差异的机制。因此，我们评估了CYP3A4 / ABCB1 mRNA 表达（聚合酶链式反应）、CYP3A4/P-gp 蛋白表达（免疫亲和-液相色谱-质谱、IA-LC-MS/MS）、CYP3A4 活性（睾酮羟基化），并考虑细胞内增加利福霉素浓度（0.01–10 µM）治疗后的药物摄取。此外，还分析了利福霉素对 CYP2C8、CYP2C9 和 CYP2C19 蛋白水平的影响 (IA-LC-MS/MS)。机制分析包括评估可能的自杀CYP3A4抑制（IC ₅₀变化测定）和药物对翻译效率的影响（无细胞发光测定）。利福布丁在肝细胞中的积累比利福平高6至15倍，但诱导CYP3A4 mRNA的能力与利福平相当（例如利福平61倍与利福布汀44倍，72小时）。例如，虽然利福平显着增强了蛋白质（10 µM：21 倍）和活性水平（53 倍），但与利福平相比，利福布丁仅略微增加了 CYP3A4 蛋白表达（10 µM：3.3 倍）或活性（11 倍）。 72 小时。两种利福霉素同样影响其他消除蛋白的表达。实验排除了特定利福布丁代谢物潜在的 CYP3A4 自杀抑制或核糖体功能破坏。总之，缺乏蛋白质增强作用可以解释利福布丁体内诱导相关药物相互作用风险较弱的原因。